JHSCI 2014 v4 I1 April

UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES
UNIVERZITET U SARAJEVU FAKULTET ZDRAVSTVENIH STUDIJA
Journal of Health Sciences

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Mirza Dili (BiH)

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Demal Pecar (BiH)
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Table of contents:
RESEARCH ARTICLES
Lymphangiogenesis in breast carcinoma is present but insufficient for metastatic spread
MIRSAD DORI, SUADA KUSKUNOVI-VLAHOVLJAK, SVJETLANA RADOVI, AJNA HUKI,
MIRSAD BABI, EDINA LAZOVI-SALIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
Correlation of Body Mass Index and Waist Hip Ratio with lipid
and hormone profile in women in menopausal transition
LEJLA MEALI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-18
Antibiotic combinatorial approach utilized against extended spectrum betalactamase (ESBL) bacteria isolates from Enugu, South Eastern Nigeria
RUTH A. AFUNWA, DAMIAN C. ODIMEGWU, ROMANUS I. IROHA, CHARLES O. ESIMONE . . . . . . . . . . 19-25
Influence of cigarette smoking on bone mineral density in postmenopausal
women with estrogen deficiency in menstrual history
AMILA KAPETANOVI, DIJANA AVDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26-30
Characteristics of Patients Involved in Psychotherapy in Bosnia and Herzegovina
SABINA ALISPAHI, ENEDINA HASANBEGOVI-ANI, ENITA TUCE,
NINA HADIAHMETOVI, ANETA SANDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31-35
Evaluation of tumor marker HE4 assay on the Elecsys 2010 analyzer
JOZO ORI, LEJLA HASANBEGOVI, ALEKSANDAR BODULOVI, JASMINKA MUJI . . . . . . . . . . . . . . 36-39
Comparison of 3D Maximum intensity projection (MIP) reconstruction and 2D T2 HalfFourier Acquisition Single-Shot Turbo Spin-Echo (HASTE) sequence in magnetic resonance
cholangiopancreatography
FUAD JULARDIJA, ADNAN EHI, DAMIR JAGANJAC, ESAD VOLODER,
SREKO MAURA, DUNJA VRCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40-44
Electroneurographic parameters in patients with metabolic syndrome
SULJO KUNI, EMIR TUPKOVI, MEDIHA NII, SEMIHA SALIHOVI. . . . . . . . . . . . . . . . . . . . . . . . . . . . 45-49
The incidence of vascular complications after coronary angiography:
evaluation of results and risk factors
HARIS VRANI, ILIRIJANA HAXHIBEQIRI-KARABDI, AMEL HADIMEHMEDAGI . . . . . . . . . . . . . . . . . . 50-54
The effect of the infection Clostridium difficile on the rehabilitation
EDINA TANOVIC, HARIS TANOVIC, ALDIJANA KADIC, DEVAD VRABAC,
SENAD SELIMOVI, DRAGAN KOSTI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55-58
CASE REPORTS
Cardiac aspects of DiGeorge syndrome: a report of two cases with molecular analysis
SENKA MESIHOVI DINAREVI, EMINA VUKAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59-62
Antiphospholipid syndrome associated with non-infective

mitral valve endocarditis: a case report
DRAGAN STEVANOVI, DENIS MAKI, ELVIRA DAMBASOVI,
AMIR EHAJI, FARUK USTOVI, NIJAZ TUCAKOVI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63-67
Bilateral Epididymal Cyst in 14 year-old Boy: a case report
YIIT AKIN, MEHMET SARAC, ISIL BASARA, SELCUK YUCEL, AHMET KAZEZ . . . . . . . . . . . . . . . . . . . . . 68-71
INSTRUCTIONS TO AUTHORS
Instructions and guidelines to authors for the preparation and submission of manuscripts
in the Journal of Health Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72-75
Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11
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RESEARCH ARTICLE
Open Access
Lymphangiogenesis in breast carcinoma is present

but insufficient for metastatic spread
Mirsad Dori*, Suada Kuskunovi-Vlahovljak, Svjetlana Radovi, Ajna Huki,
Mirsad Babi, Edina Lazovi-Salin
Institute of Pathology, Faculty of Medicine, University of Sarajevo, ekalua 90, 71000 Sarajevo, Bosnia and Herzegovina
ABSTRACT
Introduction: The lymphatic vasculature is an important route for the metastatic spread of human cancer.
However, the extent to which this depends on lymphangiogenesis or on invasion of existing lymph vessels remains controversial. The goal of this study was to investigate the existence of lymphangiogenesis in
invasive breast carcinoma: by measuring the lymphatic vessels density (LVD) and lymphatic endothelial cell
proliferation (LECP) and their correlation with various prognostic parameters in breast cancer, including
lymphovascular invasion (LVI).
Methods: Lymphatic vessels density was investigated in 75 specimens of invasive breast carcinoma by
immunostaining for D2-40 using the Chalkley counting method. Endothelial proliferation in lymphatic
vessels was analyzed by dual-color immunohistochemistry with D2-40 and Ki-67.
Results: Decrease of intra and peritumoral LVD in invasive breast carcinoma compared to fibrocystic
breast disease was detected (p=0.002). Lymphatic endothelial cell proliferation was significantly higher
in invasive breast cancer (p=0.008) than in the fibrocystic breast disease. LECP showed a correlation with
histological grade of the tumor (p=0.05). Involvement of axillary lymph nodes with metastatic tissue was
in strong correlation only with existence of lymphatic vascular invasion (p=0.0001).
Conclusion: These results suggest that development of breast cancer promotes proliferation of lymphatic
endothelial cells whose level correlates with histological grade of tumor, but in a scope that is insufficient
to follow growth of tumor tissue that invades them and destruct them. This might explain the decrease
of lymphatic vessels density.
Keywords: breast carcinoma; D2-40; Ki-67; lymphangiogenesis
INTRODUCTION
The major cause of death from breast cancer is dissemination of the primary tumor leading to forma*Corresponding author: Mirsad Dori
Institute of Pathology, Faculty of Medicine, University of Sarajevo,
ekalua 90, 71000 Sarajevo, Bosnia and Herzegovina
Phone: 061/220-428
E-mail: mdoric@lsinter.net
Submitted March 27 2014/Accepted April 18 2014
UNIVERSITY OF SARAJEVO
FACULTY OF HEALTH STUDIES
tion of metastases. Spread to axillary lymph nodes

is often the first step of generalization (1). Tumorassociated lymphatic vessels are considered to be the
main route of tumor cells to axillary lymph nodes
(2). Recently, lymphangiogenesis, the formation of
new lymphatic vessels, has become a new research
frontier in tumor metastasis since the discovery of
the two major lymphatic vessel growth factors-C
(VEGF-C) and -D (VEGF-D), as well as reliable
2014 Mirsad Dori et al.; licensee University of Sarajevo - Faculty of Health Studies. This is
an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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TABLE 1. Clinicopathological characteristics of the studied

cases
lymphatic markers that have allowed observation

and isolation of lymphatic endothelium (3).
Specific lymphatic endothelial markers have become
available, making analysis of lymphatics in cancer
possible. D2-40 antibody was reported to detect a
fixation-resistant epitope on a 40 kDa O-linked sialoglycoprotein expressed in lymphatic endothelium
but not blood vessels, and can be used to assess lymphangiogenesis specifically in conventionally processed formalin-fixed and paraffin-embedded tissue
specimens (4,5).
Although proliferating lymphatics, that promote
nodal metastasis, have been demonstrated in experimental breast tumors, it has yet to be determined
whether the same phenomena occurs in spontaneous
human breast cancers. The existence of lymphangiogenesis in breast carcinoma is one of the most controversial areas in the breast cancer literature.
The aim of this study was to investigate existence
of lymphangiogenesis in invasive breast carcinoma:
by measuring lymphatic vessels density (LVD) and
lymphatic endothelial cell proliferation (LECP),
and their correlation with various prognostic parameters in breast cancer, including lymphovascular
invasion (LVI).
Variable
Tumor size (AJCC)
pT1
pT2
pT3
Tumor type
Ductal (NOS)
Lobular
Medullary
Neuroendocrine
Mucinous
Tubular
Grade
1
2
3
ER status
Positive
Negative
PR status
Positive
Negative
HER2 status
0
1+
2+
3+
Ki-67
Negative
Positive
bcl2
Negative
Positive
p53
Negative
Positive
Nodal status
Negative
Positive
Lymphovascular invasion (LVI)
Negative
Positive
METHODS
Clinico-pathological data
Seventy-five cases of invasive carcinoma (IC) of the

breast were identified by searching the database of
the Institute of Pathology, Medical faculty of Sarajevo, Bosnia and Herzegovina. All patients with IC
of the breast underwent partial or total mastectomy
with axillary lymph node dissection. No neoadjuvant chemotherapy or radiotherapy was administered before the surgical treatment. Ten samples
fibrocystic breast tissue were used as a control group.
The patient age at the time of surgery ranged from
37 to 87 years, (mean 59.43 years). Clinicopathological characteristics of the studied cases are shown
in Table 1.
Immunohistochemical staining
Double-labeling immunohistochemical staining. Fourmicron-thick paraffin sections were taken on charged
Cases
(%)
41
32
2
54.7
42.7
2.7
57
8
4
3
2
1
76
10.7
5.3
4
2.7
1.3
23
33
19
30.7
44
25.3
48
27
64.2
35.8
45
30
54.7
45.3
43
13
3
16
50.6
15.3
3.5
18.8
3
72
4
96
41
34
54.7
45.3
41
34
54.7
45.3
68
7
90.7
9.3
46
29
61.3
38.7
slides, deparaffinized in xylene, and hydrated with

graded alcohols and distilled water. To detect divid5
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ing lymph vessel endothelial cells, a double immunostaining for D2-40 and the proliferation marker
Ki-67 was done. First, a monoclonal antibody directed at Ki-67 (Dako Cytomation; dilution 1:60,
klon MIB 1) was applied to the rehydrated paraffin sections for 15 minutes after antigen retrieval in
TBS (tris)-EDTA buffer (pH 9.0) at 95C. Sections
were incubated with EnVision+ Dual Link solution before development with diaminobenzidine
(Dako Cytomation). Sections were then stained
with the D2-40 antibody (Dako Cytomation; dilution 1:100) for 60 minutes. EnVision System alkaline phosphatase and Fast Red chromogen (Dako
Cytomation) were used to visualize binding of this
second antibody.
Immunostaining for ER, PR, HER2, p53, bcl-2 and
Ki-67. The primary antibody against the estrogen
receptor was performed in humidity chamber in
EDTA buffer (pH 9) for 40 min. (clone 1D5, Dako
Cytomation; dilution 1:30). The protocols for staining PR, Ki-67, and p53 included a microwave antigen retrieval step, 3 times for 5 minutes: anti-PR
(clone PgR, Dako Cytomation; dilution 1:30), antiKi-67 (clon MIB-1, Dako Cytomation; dilution
1:10), anti-p53 (clone DO-7, Dako Cytomation;
dilution 1:5). Citrate buffer (pH 6) was used for
anti-bcl-2 oncoprotein (clone 124, Dako Cytomation; dilution 1:40). The working system used was
LSAB2 (labelled streptavidin biotin) HRP and diaminobenzidine (DAB) was the chromogen used for
reaction visualization.
Antigen retrieval for HER2 using HercepTest was
performed following the manufacturer's protocol
(Dako Cytomation).
Immunohistochemistry evaluation. The percentage of
tumor cells with unequivocal nuclear staining for
estrogen receptor (ER), progesterone receptor (PR),
p53, Ki-67 (MIB-1), was recorded semiquantitatively (0, no staining; 1, <10%; 2, 11-33%; 3, 3466%; 4, 67-100%). For Bcl-2 the intensity of cytoplasmic staining (0-4) and the percentage of positive
cells were recorded. A cutoff value was applied to
each marker to indicate positive or negative staining.
A threshold of 1% for ER, PR, and 10% for p53
and Ki-67 (MIB-1) was used and score of 3+ for
HER2. For Bcl-2, there was little difference between
the different measures of positivity (i.e., percentage
of positive cells versus intensity of staining) and a

cutoff value of 10% was used.
Membranous staining was scored for HER2 according to the HercepTest (Dako) as follows: 0, no staining or faint incomplete staining in <10% cells; 1,
faint incomplete staining in >10% cells; 2, weak to
moderate complete staining in >10% cells; 3, strong
complete staining in >10% cells. Cases scored as 2+
were considered equivocal, and retested using chromogen in situ hybridization (CISH).
Lymphatic vessels quantification
Sections stained with D2-40 were used for the evaluation of LVD using the Chalkley counting method.
Each section was first scanned at low-power magnification (40) to select the most vascularized areas;
three hot spots were selected. Two authors first examined 10% of specimens to agree on which fields
to be used as hot spots. A 25-point Chalkley eyepiece graticule was applied to each hot spot and oriented to permit the maximum number of points to
hit on, or within the areas of immunohistochemically highlighted microvessel using 200 magnification.
A Chalkley count for an individual tumor was taken
as the mean value of the three graticule counts (6).
Assessment of lymphatic endothelial cell proliferation
The fractions of proliferating lymphatic endothelial

cells (LECP%) were calculated in each hotspot as
the number of lymphatic endothelial cells with Ki67stained nuclei per 100 lymphatic endothelial cells.
Cases in which the average number of lymphatic
endothelial cells in three hotspots was <10 were excluded for statistical analysis.
STATISTICAL ANALYSIS
Statistical analysis was performed using SPSS for

Windows (version 13; SPSS, Chicago, Il, USA).
Two-tailed unpaired t test was performed to identify the differences between two groups. One-way
ANOVA test was performed for comparing the
groups that were used for the t test. The correlations
between the variables were assessed by the Spearman
rank sum test. P values <0.05 were considered as statistically significant.
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FIGURE 1. (A) The lymphatic vessels were elongated and linear, dispersed around the lobules in the interlobular stroma (D2-40
x 100). (B) D2-40/Ki-67 positive peritumoural lymphatic vessels (x 100)
RESULTS
ing vessels entrapped within the tumor (Figure

1B). Blood vessels within the tumor mass were not
associated with perivascular lymphatic vessels. The
density of intratumoral and peritumoral lymphatic
vessels in invasive carcinoma was significantly lower
(p=0.002) in comparison to fibrocystic disease (Figure 2). Conversely, density of peritumoral lymphatic
vessels was significantly higher in comparison to intratumoral lymphatic vessel density (p=0.0001).
Benign breast samples
In these 10 cases, the lymphatic vessels were dispersed around the lobules in the interlobular stroma,
adipose tissue, and adjacent to blood vessels. These
vessels were elongated and linear in most areas and
tortuous focally. Lymphatic vessels were not identified within the intralobular stroma (Figure 1A).
Lymphatic endothelial cell proliferation was observed in 3 of 10 cases.
Invasive carcinoma
Lymphatic vessels were identified within invasive

tumors except in areas adjacent to preexisting ducts
and lobules; the latter were interpreted as preexist-
FIGURE 2. Decrease of intra and peritumoral LVD in invasive breast carcinoma compared to fibrocystic breast disease
(p=0.002).
FIGURE 3. LECP show correlation with histological grade of

the tumor (p=0.05).
7
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rent study, lymphangiogenesis was assessed by both

these methods. We did not find an increased LVD
in breast cancer. Dual immunohistochemistry using D2-40 and Ki-67 showed expression of Ki-67
in lymphatic endothelial cells indicating proliferation. The results of the present study confirmed Ki67-positive nuclei in a proportion of lymph vessel
endothelial cells, suggesting that there is indeed
lymphangiogenesis in breast cancer, the most compelling evidence being the presence of proliferating
lymphatic endothelial cells.
Vleugel et al. (8) and Williams et al. (9) failed to
detect lymphangiogenesis in breast cancer using
LYVE-1 as a marker for lymphatic vessels. Williams
et al. (9) did not find dividing lymphatic vessels
within the tumor in their analysis of 75 cases of invasive ductal and lobular breast cancer using dual
immunohistochemistry for LYVE-1 and Ki-67. In
contrast, Choi et al. (10) studied LVD without differentiating between intratumoral and peritumoral
lymphatic vessels in breast cancer using D2-40 antibody. They found that increased LVD correlated
with several prognostic factors, including lymph
node metastasis. Bono et al. (11) found intratumoral LYVE-1-positive vessels in only 12% of cases, in
contrast of finding such vessels in a peritumoral location in 94% of cases. Increased number of peritumoral lymphatic vessels were associated with a poor
outcome. These results are similar to those of Nakamura et al. (12) who found increased flt-4-positive
vessel density to be associated with VEGF-D expression, positive nodal status, and poor prognosis.
Detection of dividing lymphatic endothelial cells
became central to the current controversy on
whether lymphangiogenesis occurs in breast cancer
and whether lymphatic metastasis occurs through
pre-existing or newly-formed lymphatic vessels
(13-15). This controversy exists because neither the
mere presence of pro-lymphangiogenic factors nor
frequent incidence of lymph node metastasis constitutes a proof of tumor-induced, actively ongoing
formation of new lymphatic vessels. Resolving this
question has been sought by double staining using
antibodies to specific lymphatic markers (LYVE-1
or D2-40) combined with antibodies to proliferative markers such Ki-67 (MIB-1) or PCNA. As a result, interpretation of overlapped lymphatic marker/
Ki-67 positivity might depend on whether the posi-
FIGURE 4. Axillary lymph node metastasis showed strong

correlation only with existence of lymphatic vascular invasion
(p=0.0001).
Lymphatic endothelial cell proliferation was observed in the fibrocystic breast disease (in 3 of 10
cases) but was significantly higher (p=0.008) in
invasive breast cancer. LECP% showed correlation
(p=0.05) with histological grade of the tumor (Figure 3). Significant correlation was not found between lymphatic vascular density and lymphatic endothelial cell proliferation. Involvement of axillary
lymph nodes with metastatic tissue showed strong
correlation (p=0.0001) only with existence of lymphatic vascular invasion (Figure 4).
DISCUSSION
The invasion and metastasis of tumor cells are important biological features of neoplasm and the main
cause for poor prognosis and death (7). Axillary
lymph node status at time of diagnosis is the most
significant and durable prognostic factor in breast
cancer patients (1). However, the extent to which
this depends on lymphangiogenesis or on invasion
of existing lymph vessels remains controversial.
Lymphangiogenesis may be assessed either by LVD
or lymphatic endothelial proliferation. In the cur8
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tive cells are seen to be proliferating LEC, or dividing tumor cells that had invaded lymphatic vessels.
Additional challenges in detection of proliferating
LEC are: (a) a relatively low rate of vessel formation
in well-established tumors; (b) a lower density and
heterogeneity of tumor lymphatics compared with
tumor blood vessels; and (c) variability in sprouting
of new vessels at different points along the parental
lymphatic vessel (16), with the latter being undetectable in two-dimensional evaluation. Moreover,
the formation of new lymphatic vessels might not
require endothelial mitotic division if they originate
from circulating progenitors or non-endothelial
cells via trans-differentiation (17).
Given these technical and biological limitations, it is
not surprising that several studies failed to detect Ki67 or PCNA markers on LYVE-1 or D2-40-labeled
structures (13-15). However, evidence from several
research groups also supports tumor-induced lymphangiogenesis and shows its clinical relevance to
lymphatic metastasis. For instance, double Ki-67/
podoplanin staining of a large panel (N= 177) of
invasive breast carcinomas determined that 29%
of specimens displayed Ki-67 positive nuclei in
2.2% of intratumoral, peritumoral and peripheral
lymphatics (18). Frequency of positive nuclei was
strongly associated with a high lymphatic density
(p = 0.001), LN metastasis and survival (18). An
independent study detected a similar fraction of
proliferating LEC (LECP%) in peritumoral lymphatics also identified LECP% as an independent
prognostic factor for LN metastasis (19). Studies
that compared LECP% in inflammatory and noninflammatory breast cancers found that the former
have both a higher incidence of Ki-67 positive lymphatics (80% vs. 50%) and an increased median
LECP% (20, 21). Active lymphangiogenesis was
also detected in positive sentinel LN (22, 23) that
displayed a significantly higher median LECP% (p
< 0.001) than uninvolved LN (23). Moreover, high
frequency of Ki-67-labeled lymphatics in positive
sLN was strongly associated (p = 0.01) with axillary
metastasis (22), supporting the contention that tumor-induced lymphangiogenesis promotes dissemination from both the primary tumor and secondary
metastatic sites. Nevertheless, with the exception
of very active lymphangiogenesis in inflammatory
breast cancer (20,21), a relatively low fraction of di-
viding lymphatic endothelial cells (26%) and some

discrepancy between high %LVI and low %LECP
in other breast cancer types suggest that both new
and existing lymphatic vessels partake in lymphatic
metastasis (24).
In contrast to challenges mentioned earlier in the
detection of dividing LEC, enumerating lymphatic
vessels seemed initially a straightforward measure
of lymphangiogenesis. To assess tumor vascularity,
there are several methods including counting the
number of immunohistochemically stained microvessels in vascular hot spots, grading of vascular,
using image analysis systems (25) and applying the
Chalkley grid. The Chalkley count technique was
recommended in an international consensus report
because it is considered to be a simple and acceptable procedure for daily clinical use and produced
lower inter-observer variability compared to the
more frequently used conventional microvessel density method (26). LVD has been studied in a number of human cancers, including melanoma (27)
and prostate (28). As a result, findings and interpretations from the studies that focused on infrequently
occurring intratumoral lymphatic vessels (14), or
those that compared a heterogeneous LVD pattern
to more orderly tumor blood vessel distribution
(13), fueled the debate whether lymphangiogenesis
exists in breast cancer (13-15). Additional complexity arises from the fact that, in contrast to blood vessels, lymphatic vessels support spread of metastatic
cells, but not tumor cell proliferation and expansion
of the tumor mass. Kanngurn et al. (29) showed
that microvessel density (MVD) Chalkley but not
the LVD Chalkley count can be a predictive factor
for axillary lymph node metastasis in breast carcinoma. Therefore, subtle increases in LVD might be
missed in tumor sections set aside for immunohistochemical analysis, although they might suffice for
tumor dissemination in a patient. All methods used
in study of Niemiec et al. (30) for assessment of lymphangiogenesis (LVD, DLV, LVD/MVD) were correlated to each other and to parameters indicating
aggressive tumor behavior (high grade, TNP, HER2
subtype, basal marker expression), hence they might
be used equivalently.
The main evidence supporting the claim that lymphangiogenesis does not exist in tumors is detection
of decreased LVD or absence of intratumoral lym9
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Pathol. 2003;200:195206.
phatic vessels (LV) compared with normal breast tissue (13-15). The same studies, however, reported a
significant increase (p=0.0001) in peritumoral LVD
(13,15) with some lymphatic vessels containing tumor emboli (13). There is a wide range of opinions
with regard to a prognostic value of intratumoral
LVD. However, a consensus seems to exist with regard to increased density of peritumoral lymphatic
vessels that might be sufficient for tumor cell transit
to lymph node even in the absence of intratumoral
lymphatics.
(10) Choi WW, Lewis MM, Lawson D, et al. Angiogenic and lymphangiogenic
microvessel density in breast carcinoma: correlation with clinicopathologic
parameters and VEGF-family gene expression. Mod Pathol. 2005; 18:143
152.
(11) Bono P, Wasenius VM, Heikkila P, et al. High LYVE-1-positive lymphatic
vessel numbers are associated with poor outcome in breast cancer. Clin
Cancer Res. 2004;10:71447149.
(12) Nakamura Y, Yasuoka H, Tsujimoto M, et al. Flt-4-positive vessel density
correlates with vascular endothelial growth factor-d expression, nodal status, and prognosis in breast cancer. Clin Cancer Res. 2003;9:53135317.
(13) Agarwal B, Saxena R, Morimiya A, Mehrotra A, Badve S. Lymphangiogenesis does not occur in breast cancer. Am J Surg Pathol. 2005; 29:1449
1455.
(14) Vleugel MM, Bos R, van der GP, Greijer A, Shvarts E, Stel A.H.V, W.E. van
der, PJ. van Diest. Lack of lymphangiogenesis during breast carcinogenesis. J Clin Pathol. 2004; 57:746751.
CONCLUSION
(15) Van der Schaft DW, Pauwels P, Hulsmans S, Zimmermann M, van de PollFranse LV, Griffioen AW. Absence of lymphangiogenesis in ductal breast
cancer at the primary tumor site. Cancer Lett. 2007;254:128136.
The findings from this study show that lymphangiogenesis in breast cancers, measured by lymphatic
endothelial cell proliferation is present, but measured by lymphatic vascular density is absent. These
results suggest that development of cancer tissue in
breast promotes proliferation of lymphatic endothelial cells whose level correlates with histological
grade of tumor, but in a scope that is insufficient
to follow growth of tumor tissue that invades them
and destruct them. This might explain decrease of
lymphatic vessels density.
(16) Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol. 2007; 8:464478.
(17) Maruyama K, Ii M, Cursiefen C, Jackson H, Keino DG, Tomita M, Van Rooijen N, Takenaka H, DAmore PA, Stein-Streilein J, Losordo DW, Streilein
JW. Inflammation-induced lymphangiogenesis in the cornea arises from
CD11b-positive macrophages. J Clin Invest.2005; 115:23632372.
(18) Mohammed RA, Ellis IO, Elsheikh S, Paish EC, Martin SG. Lymphatic and
angiogenic characteristics in breast cancer: morphometric analysis and
prognostic implications. Breast Cancer Res Treat. 2008;261273.
(19) Van den Eynden GG, van der Auwera I, van Laere SJ, Trinh XB, Colpaert
CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Comparison of
molecular determinants of angiogenesis and lymphangiogenesis in lymph
node metastases and in primary tumours of patients with breast cancer. J
Pathol 2007;213:5664.
COMPETING INTERESTS
The authors declare no conflict of interest for this

study.
(20) Van der Auwera I, Van den Eynden GG, Colpaert CG, Van Laere SJ, van
Dam P, Van Marck EA, Dirix LY, Vermeulen PB. Tumor lymphangiogenesis
in inflammatory breast carcinoma: a histomorphometric study. Clin. Cancer
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Lejla Meali Journal of Health Sciences 2014;4(1):12-18
http://www.jhsci.ba

RESEARCH ARTICLE
Open Access
Correlation of Body Mass Index and Waist Hip

Ratio with lipid and hormone profile in women in
menopausal transition
Lejla Meali
Department for health protection of women and pregnant women at the public health institution Health Centre Tuzla,
Bosnia and Herzegovina
ABSTRACT
Introduction: Menopausal transition is a period characterised by psychic, somatic changes as well as
changes in reproductive capabilities of a woman. It occurs as a consequence of ovarians function termination, and pertains to the periods of different meanings: perimenopause, menopause and postmenopause.
Although there are numerous assessments of behaviour of the lipids and lipoproteins during menopausal
transition, their relation to sexual hormones and body mass is still being assessed. The aim of this study is
to determine the differences and connections between body mass index (BMI) and waist-hip ratio (WHR)
and lipid and hormone profile among the assessees in premenopause, perimenopause and postmenopause.
Methods: The assessment was done on 150 assessees divided in three groups of 50, such as: premenopause, perimenopause and postmenopause. The assessment included the following: interview, determination of BMI, WHR, and taking of blood sample and processing of hormone, lipid and lipoprotein
concentration.
Results: Based on the obtained results, it may not be concluded that BMI has a positive correlation with
cholesterol and VLDL concentration in postmenopause, positive correlation with apo A in perimenopause
and postmenopause, and positive correlation with Lp (a) and apo B in premenopause and perimenopause,
while negative correlation with HDL and estradiol concentrations in premenopause. WHR has negative
correlation with HDL concentration in premenopause and perimenopause, and a negative correlation with
estradiol concentrations in premenopause.
Keywords: menopause, lipids, hormones, body mass index, waist-hip ratio
INTRODUCTION
*Corresponding author: Lejla Meali
Department for health protection of women and pregnant
women, Health Centre Tuzla, Albina Herljevia 1, 75000 Tuzla,
Bosnia and Herzegovina; Phone: +387 61 146 698
E-mail: mesaliclejla@gmail.com
Submitted December 18, 2013/Accepted February 3, 2014
Menopausal transition is a period in life of a woman,

with undefined beginning and duration, but with
well-known changes in female organism. Menopausal transition is a period characterized by psychic,
somatic and reproductive changes of capabilities at
2014 Lejla Meali; licensee University of Sarajevo - Faculty of Health Studies. This is an
Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
http://www.jhsci.ba
women (1). Hormonal profile is changing during

menopausal transition. Increased level of folliclestimulating hormone (FSH) stimulates follicles to
grow, but those follicles mainly fail to reach the final
growth and maturity, which results with frequent
anovulation. Progesterone production is 60 % lower
than in reproduction period. In menopause, metabolic changes occur in different tissues and organs as
a result of changed hormonal profile (2). Fat tissue is
not only a passive fat depot reflecting the energy balance and thermo-regulation, but is also a significant
endocrine organ (3). The main source of estrogen in
menopause is estron generated from androstendion
in peripheral tissues. Conversion processes of androgen to estrogens in menopause are not performed in
fat tissue only, but also in central nervous system (4,
5). One of the most important fat tissue hormones
is leptin. During menopause, not only leptin, but
also decreased level of growth hormone, E2 and androgens lead to changes of lipogenesis and lipolysis
mechanisms, which lead to characteristic distribution of fat tissue in menopause (centripetal obesity).
Lipid profile during menopausal transition changes,
but all mechanisms of those changes are not clarified. One of the most important factors in that
mechanism is the change of fat tissue distribution
in postmenopause. Total cholesterol level increases
through menopausal transition, with highest values
in menopause. LDLD increases during menopausal transition, but it decreases after the menopause.
The age is significantly related to the changes in the
triglyceride, total cholesterol and LDL level, while
body mass index (BMI) is significantly related to
the changes of level of triglyceride, LDL, HDL (6).
With menopause, HDL concentration decreases
and HDL structure changes. HDL concentration
is inversely proportional to abdominal obesity level
(7). Menopause, age and increased distribution of
abdominal fat tissue are three independent and
important factors violating the lipoprotein profile
from the beginning of menopausal transition (8).
Age, BMI and menopausal status are significant indicators of the circulating lipoprotein level during
menopausal transition (9). BMI is used to calculate the body surface and estimate the body weight.
Doctors use BMI to estimate the risk of body weight
and cardiac disease. New analysis of 40 studies, published in the Lancet magazine (10), which includes
250 000 patients, shows that the patients with BMI

below 20 have higher mortality risk caused by cardiovascular diseases than the obese patients with
BMI 30-35. The assessees who smoke have somewhat lower total cholesterol and significantly lower
HDL, although there is no difference in BMI distribution between the assessees who smoke and those
who do not (11). E2 level is much lower at the obese,
premenopausal women than at the premenopausal
women of normal body weight. FSH level is also
lower at the obese postmenopausal women than at
the non-obese postmenopausal women (12). BMI
impact to E2 and FSH varies in dependence on
menopausal profile (13). Waist-hip ratio, WHR is
statistically important predictor of LDL concentration and cholesterol in relation to BMI at women in
menopause, which confirms the importance of fat
tissue distribution as a risk factor for cardiovascular
diseases at this group of women (14). Sultan and
associates (15) state that waist-hip ratio may be used
as screening for identification of postmenopausal
women with higher cardiovascular risk.
The aim of this study was to determine the differences and connection of BMI and WHR with lipid
and hormonal profile of women in premenopause,
perimenopause and postmenopause.
METHODS
This prospective, comparative study was conducted

in period November 2009 December 2010 and it
included a total of 150 assessees aged 40-55, divided
into three groups of 50 assessees each (premenopause, perimenopause and postmenopause). The
assessees comprised women volunteers who agreed
in writing to be included in the study. Each assessee was introduced with the character of the study
implemented according to the generally accepted
ethical standards for medical research. Qualification criteria for inclusion into the study were: that
the candidates do not take hormonal supplemental therapy, that they do not take medicines which
could affect the lipid profile, that they do not consume more than twenty cigarettes a day, that their
BMI does not exceed 35kg/m. Processing of assessees comprised three phases: interview, taking blood
sample, measuring BMI and WHR. For the purpose
of analysis, the vein blood sample from cubital vein
13
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TABLE 1. Main features of assessees1
was taken, after which the blood was centrifuged,

and obtained serum divided into two test tubes. The
same day, concentration of lipid, lipoprotein, FSH,
LH and E2 was determined. Concentration of the
total cholesterol, triglyceride, LDL, HDL, VLDL,
apo A, apo B and Lp (a) was also determined. Total cholesterol and triglyceride concentration was
determined using enzymatic method at SIEMENS
Dimension RxL machine. HDL and LDL were
determined at the same machine utilizing direct
homogenous method. VLDL was calculated with
formula: VLDL = total cholesterol - HDL LDL
apo A, apo B and Lp (a) values were determined
using immunoturbidimetry method at SIEMENS
Dimension RxL machine, and reagents of the
company SENTINEL were used. FSH, LH and
E2 values were determined utilizing the hemiluminiscence method. The assessees' body weight and
height were measured at the SECAscale with meter. Fattening status was assessed based on the Qeuetelet index (Devenport-Kaup modification) or BMI
where: BMI= body mass in kg/height in m. Based
on the waist measuring at the narrowest place and
hip measuring at the widest place, WHR was calculated according to the following formula: WH ratio
= waist (cm)/hip (cm).
Age
Menarche,
age
Age at last
menstruation
Reproductive
age
Group I
(n=50)
45.4 3.2
(40-52)
14.2 1.6
(11-18)
-
BMI (kg/m2)
26.3 3.7
(20.0-36.0)
WHR
0.81 0.05
(0.70-0.97)
Group I
(n=50)
47.4 2.6
(41-52)
13.8 2.0
(10-18)
47.2 2.5
(41-52)
33.4 2.7
(26-39)
26.4 3.8
(18.5-37.0)
0.79
0.08
(0.43-0.90)
Group III
P value
(n=50)
51.4 3.2
<0.001
(43-55)
14.1 1.7
0.574
(11-17)
47.9 3.3
0.262
(39-54)
33.8
0.570
(26-42)
26.9 3.4
0.640
(22.0-36.0)
0.81
0.06
0.076
(0.67-0.90)
1
Values are arithmetic median value SD as well as minimum
and maximum. Group I= assessees in premenopause; group II=
assessees in perimenopause; group III= assessees in postmenopause.
years), and the assessees in postmenopause were four

years older in average than those in perimenopause
(95% IP: 2.6-5.4 years) (Table 1). In addition, the
assessees from the second group had somewhat lower WHR than the assessees from the third group, although the difference was not statistically significant
(average difference = 0.03, 95% IP: -0.003-0.06).
There is a significant negative relation between BMI
and HDL at assessees in premenopause (Table 2).
If the regression coefficient had been standardised
to interquartile value of BMI change (IQR=5), the
difference of 0.18 mmol/L in medium HDL concentration between low and high BMI would have
been suggested. Among the assessees in postmenopause, there is a significant relation between BMI
and cholesterol, as well as between BMI and VLDL.
For interquartile value of BMI change, standard
regression coefficient shows the increase of 0.67
mmol/L in medium cholesterol concentration and
increase of 0.22 mmol/L in medium VLDL concentration. There are no proofs of any relation between other lipid and BMI parameters. Multivariate
regression analysis showed that the only important
lipid fraction affected by BMI is HDL cholesterol.
Considering the BMI value in relation to apo A,
results indicate that there is a correlation between
the groups of assessees in perimenopause and post-
Statistical analysis
To compare the numerical variables among the assesse groups we used the variance analysis (ANOVA) or Kruskal-Wallis non-parametric alternative.
To analyse relation of BMI, WHR, menarche and
reproductive age factors with lipid and hormonal
profile, we used linear regression model. Each factor
was tested in bivariate model, adjusted to the group
of assessees. Results are presented at regression coefficient with related 95% reliability interval (IP).
Statistical significance was confirmed at p<0.05.
Statistic programme PASW 18 (SPSS Inc., Chicago,
Illinois, USA) was used for data processing.
RESULTS
The assessees included in the study were aged 40 to

55, with average age 48.1 3.9. Statistically, the age
between the groups differed significantly: the assessees in perimenopause were 2 years older in average than those in premenopause (95% IP: 0.6-3.5
14
-0.104, 0.047
-1.1, 7.2
-0.057, -0.012
-0.065, 0.052
-0.022, 0.046
-0.048, 0.010
-0.034, 0.015
-4.3, 11.7
3.0
-0.035
-0.006
0.012
-0.019
-0.010
3.5
0.004
0.834
0.482
0.191
0.426
0.376
0.146
0.445
P value
-0.018
-0.048
0.016
0.017
-0.013
-0.9
2.3
Regression
coefficient
-0.050
-0.042, 0.006
-0.116, 0.019
-0.025, 0.056
-0.014, 0.048
-0.033, 0.006
-11.2, 8.2
-1.6, 6.4
-0.135, 0.034
95% IP
Group II
0.143
0.158
0.436
0.283
0.170
0.833
0.232
0.236
P value
0.020
0.070
0.043
-0.004
-0.011
0.5
0.0
Regression
coefficient
0.133
-0.009, 0.050
-0.015, 0.154
0.005, 0.080
-0.027, 0.020
-0.038, 0.016
-9.8, 11.7
-2.9, 2.8
0.032, 0.234
95% IP
Group III
0.177
0.103
0.027
0.747
0.432
0.948
0.935
0.011
P value
15
-0.768, 1.972
-0.383, 1.498
2.8, 15.3
8.9
0.005
0.381
0.239
P value
-3.3
Regression
coefficient
-0.634
-0.658
-10.7, 3.6
-2.383, 1.116
-1.988, 0.673
95% IP
Group II
Group I = assesses in premenopause; group II = assesses in perimenopause; group III = assesses in postmenopause.
FSH, IU/L
LH, IU/L
Estradiol, % decrease
to BMI unit
95% IP
Regression
coefficient
0.602
0.557
Group I
TABLE 3. Regression coefficient for hormonal profile in relation to BMI.
0.358
0.470
0.325
P value
3.8
Regression
coefficient
0.354
0.165
-4.9, 13.2
-2.344, 3.053
-1.027, 1.358
95% IP
Group III
0.399
0.793
0.781
P value
Values are arithmetic median value SD as well as minimum and maximum. Group I= assessees in premenopause; group II= assessees in perimenopause; group III= assessees in postmenopause.
Cholesterol, mmol/L
Triglyceride, % increase to
BMI unit
HDL, mmol/L
LDL, mmol/L
VLDL, mmol/L
Apo A, g/L
Apo B, g/L
Lp (a), % decrease to BMI unit
95% IP
Regression
coefficient
-0.029
Group I
TABLE 2. Regression coefficient for lipid profile in relation to BMI.
http://www.jhsci.ba
-0.204, 0.327
0.99, 1.30
-0.186, -0.023
-0.130, 0.279
-0.024, 0.207
-0.131, 0.076
-0.089, 0.082
0.84, 1.44
1.13
-0.105
0.074
0.091
-0.027
-0.004
1.10
0.474
0.013
0.468
0.119
0.595
0.929
0.070
0.645
P value
1.07
-0.089
0.022
0.026
-0.040
0.006
1.06
Regression
coefficient
-0.041
0.85, 1.36
-0.144, -0.034
-0.152, 0.195
-0.075, 0.127
-0.117, 0.037
-0.043, 0.055
0.96, 1.17
-0.255, 0.173
95% IP
Group II
16
-3.436, 6.212
-2.888, 3.796
1.05, 1.59
1.29
0.019
0.566
0.786
P value
0.93
Regression
coefficient
-3.848
-2.018
0.91, 1.29
-8.104, 0.408
-5.328, 1.292
95% IP
Group II
FSH, IU/L
LH, IU/L
Estradiol, decrease coefficient
to WHR increase for 0.05
95% IP
Regression
coefficient
1.388
0.454
Group I
TABLE 5. Regression coefficient for hormonal profile in relation to WHR.
Cholesterol, mmol/L
Triglyceride, increase coefficient to WHR increase for
0.05
HDL, mmol/L
LDL, mmol/L
VLDL, mmol/L
Apo A, g/L
Apo B, g/L
Lp (a), decrease coefficient to
WHR increase for 0.05
95% IP
Regression
coefficient
0.061
Group I
TABLE 4. Regression coefficient for lipid profile in relation to WHR, calculated for WHR change of 0.05.
0.376
0.075
0.226
P value
0.549
0.002
0.803
0.610
0.304
0.812
0.232
0.700
P value
1.13
Regression
coefficient
3.889
2.005
1.05
0.017
0.195
-0.029
-0.028
0.023
1.07
Regression
coefficient
0.183
0.88, 1.44
-3.653, 11.431
-1.315, 5.324
95% IP
Group III
0.78, 1.15
-0.069, 0.102
-0.044, 0.434
-0.140, 0.083
-0.094, 0.039
-0.053, 0.100
0.99, 1.15
-0.119, 0.484
95% IP
Group III
0.336
0.305
0.231
P value
0.729
0.692
0.107
0.605
0.403
0.543
0.100
0.229
P value

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http://www.jhsci.ba
menopause according to the first significance order

(p<0.001), while between the assessees in premenopause and postmenopause there is no significant
difference (p=0.516). Correlating BMI value in
relation to apo B, there was statistically significant
difference between the assessees in perimenopause
and premenopause (p=0.003), while between the assessees in postmenopause and premenopause there
was no statistically significant difference (p=0.367).
Obtained results regarding Lp (a) indicate the significant correlation in relation to BMI, only among
the asssessees in postmenopause and premenopause
(p<0.001). Of three analysed hormones, significant
relation with BMI is noted only for E2 at women
in premenopause (Table 3). For increase of unit in
BMI, regression coefficient suggests 8.9-pecent decrease in medium E2 concentration. There is a significant negative relation between WHR and HDL
at the assessees in premenopause and perimenopause (Table 4). At WHR change of 0.05, regression coefficient shows the difference of 0.11 mmol/L
in medium HDL concentration at the assessees in
premenopause, and the difference of 0.09 mmol/L
in medium HDL concentration at the assessees in
perimenopause. The only significant relation between WHR and hormone parameters is proven for
E2 at the assessees in premenopause (Table 5). Medium estradiol concentration decreased 1.29 times
at WHR increase for 0.05.
There are no proofs of any relation between other lipid and BMI parameters. Other studies also indicate
that women with higher BMI are exposed to the risk
of higher lipid level, although skinner women can
also have higher hormone-related LDL cholesterol
during menopausal transition (16). Duration of
postmenopause, as well as BMI in similar studies do
not show significant correlation with lipid, lipoprotein and Lp (a) concentration, while WHR shows
significant positive correlation with cholesterol,
LDL and apo B (14) concentrations. According to
Yamamoto and associates (11), there is no significant
correlation between BMI and serum Lp (a) value,
and medium Lp (a) value shows the possible trend
of increase at women over 40. This study showed
significant correlation between BMI and Lp (a) at
the assessees in postmenopause and premenopause,
which is confirmed by contemporary knowledge on
impact of higher Lp (a) to the increase of cardiovascular risk. We determined a significant negative
relation between WHR and HDL cholesterol at
the assesses in premenopause and perimenopause,
while in relation to other lipids, there were no correlations. According to Meali (14), among women
in premenopause, WHR has a significant negative
correlation with HDL and Apo lipoprotein A concentration, which, considering the role of these two
lipoproteins in occurrence of cardiovascular diseases,
confirms that even women with regular menstruations and higher WHR have the risk of cardiovascular diseases. Results of this study indicate negative correlation between WHR and estradiol at the
assesses in premenopause and negative correlation
between BMI and estradiol in premenopause. However, with increase of WHR and BMI, the estrogen
level decreases in premenopause, based on which
the women with higher cardiovascular risk may be
identified. Assessment results of the comprehensive
study conducted by the National Health and Nutrition Examination Survey among the assessees aged
35-60 in period 1999-2002 (17) show that there are
no significant differences in total cholesterol, triglyceride, HDL, LDL cholesterol levels adjusted to
the age, among menopausal periods at the group of
women with normal BMI. The difference in HDL
cholesterol values was noticed at the groups with
normal and higher BMI. In the groups of assessees
with normal BMI, LH and FSH hormones activ-
DISCUSSION
Numerous studies addressed the issue of lipid changes during menopausal transition or related to menopausal changes of endogenous hormones. Although
the numerous researches have been done on behaviour of lipids and lipoprotein during menopausal
transition, their relation with sexual hormones and
body mass is still being assessed. The average age in
time of the last menstruation among the assessees
included in this study is 48. One of the objectives of
this study was to determine the relation of BMI and
WHR with lipid and hormonal profile of assessees
in menopausal transition, and the results show that
at the assessees in postmenopause, there is a positive correlation between BMI and total cholesterol,
and BMI and VLDL cholesterol, and negative correlation between BMI and HDL in premenopause.
17
http://www.jhsci.ba
3. Yen SSC, Jaffe RB, Barbieri RL. Reproductive endocrinology. Philadelphia:

WB Saunders, 1999;191-217.
ity was statistically different than at the assessees in

premenopause and perimenopause, and between
the assessees in premenopause and postmenopause
(17). SO, according to Azizi and Ainy (18) BMI,
WHR and triglyceride value in blood does not show
the significant differences between the assessees in
premenopause, perimenopause and postmenopause.
Despite numerous studies, physiological role of Lp
(a) has not been determined. Its atrogenous influence is attributed to enormous similarity of Lp(a)
and LDL in structure, which are very cholesterol
rich. In this study, Lp (a) was the highest at the assessees in premenopause and postmenopause. Somewhat different results are presented by Kim and associates (19), who obtained lower Lp (a) values at
perimenopausal assessees than at postmenopausal
assessees.
4. Naftolin F. Brain Aromatisation of Androgens. J of Repr Med. 1994;39:257261.

5. Vujovi S. Central nervous system and sexual steroids. In: Vujovi S.
Menopause. Belgrade: Kosmos. 1998;33-39.
6. Torng PL, Su TC, Sung FC, Chien KL, Huang SC, Chow SN, Lee YT. Effects of menopause on intraindividual changes in serum lipids, blood pressure, and body weight--the Chin-Shan Community Cardiovascular Cohort
study. Atherosclerosis. 2002;161(2):409-15.
7. Lamarche B, Moorjani S, Cantin B, Dagenais GR, Lupien PJ, Despers JP.
Association of HDL2 and HDL3 subfractions with ischemic heart disease in
men. Prospective results from the Qubec cardiovascular study. Arterioscler
Thromb Vasc Biol 1997;17:1098-1105.
8. Berg G, Mesh V, Boero L, Sayegh F, Prada M, Royer M, Muzzio ML, Schreier L, Sisele N, Benanacia H. Lipid and lipoprotein profile in menopausal
transition. Effects of hormones, age and fat distribution. Horm Metab Res
2004; 36 (4):215-220.
9. Hall G, Collins A, Csemiczky G, Landgren BM. Lipoproteins and BMI: a
comparison between women during transition to menopause and regularly
menstruating healthy women. Maturitas 2002; 41(3):177-185.
10. Romero-Corral A, Montori VM, Somers VK, Korinek J, Thomas RJ, Allison
TG, Mookadam F, Lopez-Jimenez F. Association of bodyweight with total
mortality and with cardiovascular events in coronary artery disease: a systematic review of cohort studies. The Lancet 2006;368:666-678.
CONCLUSION
11. Yamamoto A, Horibe H, Mabuchi H, Kita T, Matsuzawa Y, Saito Y, Nakaya

N, Fujioka T, Tenba H, Kawaguchi A, Nakamura H, Goto Y. Analysis of
serum lipid levels in Japanese men and women according to body mass
index. Increase in risk of atherosclerosis in postmenopausal women.
Research Group on Serum Lipid Survey 1990 in Japan. Atherosclerosis.
1999; 144 (1):55-73.
BMI has a positive correlation with cholesterol concentration and VLDLD in postmenopause, positive
correlation with apo A in perimenopause and postmenopause, and positive correlation with Lp (a) and
apo B in premenopause and perimenopause, while
with HDL and estradiol concentration it has a negative correlation in premenopause.
WHR has negative correlation with HDL concentration in premenopause and perimenopause, and
negative correlation with estradiol concentrations
in premenopause. However, with increase of WHR
and BMI, the estrogen level in premenopause decreases, based on which the women with higher
cardio-vascular risks may be identified.
12. Freeman EW, Sammel MD, Lin H, Gracia CR. Obesity and reproductive hormone levels in the transition to menopause. Menopause 2010;17(4):71826.
13. Randolph JF Jr, Sowers M, Bondarenko I, Gold EB, Greendale GA, Bromberger JT, Brockwell SE, Matthews KA. The relationship of longitudinal
change in reproductive hormones and vasomotor symptoms during the
menopausal transition. J Clin Endocrinol Metab 2005;90(11):6106-6112.
14. Meali L. Lipid profile of women in menopause. Master study paper. Faculty of medicine, University in Tuzla. 2005;34-58.
15. Sultan N, Nawaz M, Sultan A, Fayaz M. Waist hip ratio as an index for
identifying women with raised TC/HDL ratios. J Ayub Med Coll Abbottabad.,
2004;16(1):38-41.
16. Derby CA, Crawford SL, Pasternak RC, Sowers M, Sternfeld B, Matthews
KA. Lipid Changes During the Menopause Transition in Relation to Age
and Weight. The Study of Women's Health Across the Nation. American J
of Epidemiol 2002;169(11):1352-1361.
COMPETING INTERESTS
REFERENCES
17. Wiacek M, Hagner W, Zubrzycki IZ. Measures of menopause driven differences in levels of blood lipids, follicle-stimulating hormone, and luteinizing
hormone in women aged 35 to 60 years: National Health and Nutrition Examination Survey III and National Health and Nutrition Examination Survey
1999-2002 study. Menopause 2011;18(1):60-66.
1. Petrovi L, Korkoci D, Radukanovi V, Pusloji J. Menopausal transition

and how to live it through in a quality manner. Medical Gazette of Timocki,
2006;71-80.
18. Azizi F, Ainy E. Coronary heart disease risk factors and menopause: a study
in 1980 Tehranian women, the Tehran Lipid and Glucose Study. Climacteric the journal of the International Menopause Society. 2003;6(4):330-336.
2. Dopsaj V, umarac Z. Changes of biochemical parameters in menopause.

Pharmacy archives. 2005;55 (2):76-90.
19. Kim CJ, Kim TH, Ryu WS, Ryoo UH. Influence of menopause on high density lipoprotein-cholesterol and lipids. J Korean Med Sci 2000;15(4):380-6.
The author declares no comflict of interest.
18
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Ruth A. Afunwa et al. Journal of Health Sciences 2014;4(1):19-25

RESEARCH ARTICLE
Open Access
Antibiotic combinatorial approach utilized against

extended spectrum beta-lactamase (ESBL) bacteria
isolates from Enugu, South Eastern Nigeria
Ruth A. Afunwa1, Damian C. Odimegwu1,2*, Romanus I. Iroha3, Charles O. Esimone4
1
Division of Pharmaceutical Microbiology, Department of Pharmaceutics, University of Nigeria, Nsukka, 410001, Enugu
State, Nigeria. 2Department of Molecular and Medical Virology, Ruhr University Bochum Universitt Strae 150, Bochum
44780 Germany. 3Department of Applied Microbiology, Ebonyi State University,PMB 053, Abakaliki, Ebonyi State, Nigeria.
4
Department of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe University Awka PMB 5025, Awka,
Anambra State, Nigeria.
ABSTRACT
Introduction: Antibiotic options in the treatment of extended spectrum beta-lactamase (ESBL) producing
bacteria are very limited. The purpose of this study was to analyze several commonly applied antibiotics in
quite various novel combinations for use against ESBL-producing bacteria isolates.
Methods: Total of 460 samples of urine, throat and anal swab were collected from volunteers and patients from nursery, primary and secondary schools and from other individuals in the community. Hospital
and community isolates comprised of 65% and 35% respectively. The identification and characterization
of the isolates were done by standard culturing and in vitro antibiotic sensitivity procedures.
Results: The antibiotic combination studies showed that the combination of gentamicin with the other
antibiotics had predominantly synergistic effects. The percentage synergistic effect for the combinations
of gentamicin/pefloxacin was 69%, gentamicin/[Amoxicillin and clavulanic acid] 72%, gentamicin/ceftriaxone 68%, gentamicin/cefuroxime 81.9%, and gentamicin/ciprofloxacin 80.6%, against the community
and hospital derived ESBL producing organisms of both Enterobacteriaceae and Pseudomonas species.
Conclusion: Good antimicrobial monitoring exercise and corresponding antimicrobial screening activities
should work towards a dynamic approach to generate effective treatment options using combination
therapy.
Keywords: Enterobacteriaceae, Pseudomonas aeruginosa, Extended spectrum beta-lactamase (ESBL),
Plasmid.
INTRODUCTION
*Corresponding author: Damian C. Odimegwu
Division of Pharmaceutical Microbiology, Department of Pharmaceutics, University of Nigeria, Nsukka, 410001, Enugu State, Nigeria
Department of Molecular and Medical Virology, Ruhr University
Bochum Universitt Strae 150, Bochum 44780 Germany
Phone: +4915212858200; E-mail: nonsodimegwu@yahoo.co.uk
Several community-acquired pathogens that commonly cause diarrhoea have been found to be ESBL
producers. In the last 3 years there have been reports
of true community-acquired infection or colonization with ESBL-producing Escherichia coli from
Submitted November 8th 2014/Accepted April 15th 2014

2014 Ruth A. Afunwa et al.; licensee University of Sarajevo - Faculty of Health Studies. This
is an Open Access article distributed under the terms of the Creative Commons Attribution
http://www.jhsci.ba
(NONA), and Reego Laboratories, Enugu. Characterization of isolates was according to recommended
standard technique by the National Committee for
Clinical Laboratory Standard (NCCLS).
Spain, Israel, the United Kingdom, Canada, and

Tanzania (1-4). Typically, patients have developed
urinary tract infection with CTX-M-producing
Escherichia coli. Some urinary tract infections have
been associated with bacteraemia. The majority
of isolates have been resistant to commonly used
first-line agents for urinary tract infection such as
trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and ceftriaxone. The cause of this sudden
upsurge in community-acquired infections with
ESBL-producing organisms is not yet clear, but associations with foodstuffs, animal consumption of
antibiotics, and frequent patient contact with health
care facilities need to be explored. The ESBL production status of the isolates were previously established through the double disc synergy test (DDST)
method involving the use of a combination disc
(Amoxicillin 20 g and Clavulanic acid 10 g)
placed at the centre of the Petri dish and antibiotics
(Ciprofloxacin 30 g and Cefuroxime 30 g) placed
15 mm apart on both sides of the plates (5). Also, in
our report we could establish their community or
nosocomial origins (5).
The purpose of this present work was to study some
commonly applied antibiotic in quite various novel
combinations for use against ESBL-producing bacteria isolates. In this paper, we present the result of
our antibiotic susceptibility studies of the ESBL isolates to a cocktail of antibiotics to establish a very
realistic antibiogram that may prove useful in clinical practice. The synergies that may exist between
the antibiotics involved in each combination were
analyzed.
Culture Media and Reagents
Nutrient broth (Oxoid, England), mannitol salt agar

(Oxoid, England) nutrient agar (Fluka Spain) and
peptone water were used. Sucrose, mannitol, Gram
Staining reagents, buffer solution, Tris-ethylenediamine tetra- acetic acid sodium sulfate (TENS),
sodium acetate, peptone, Ethidium bromide and
Bromo phenol blue were all analar grade reagents.
Antibiotic Discs
Antibiotic discs used were obtained from Oxoid

(England) and they include ceftriaxone (30g),
Clindamycin (30g), Ciprofloxacin (30g), Cefuroxime (30g), Augmentin(Amoxicillin and clavulanic acid) (30g), Gentamicin (30g), Pefloxacin (30g), Imipenem (30g), Cefotaxime (30g),
Ceftazidime (30g), Tetracycline (30g).
Antibiotic Sensitivity test
Antibiotic sensitivity of the isolates was determined

using previously established procedure (6). Briefly,
the isolates were cultured in nutrient broth at 370C
for 24 h. Two loopfuls of the suspension of each
isolate were inoculated into 20ml of sterile molten
agar in 10 cm diameter Petri dishes and mixed. The
plates were allowed to set and the antibiotic Sensitivity discs were aseptically placed on their surfaces.
The plates were incubated at 370C for 24 h and the
resultant inhibition zone diameters (IZDs) were
measured and recorded.
METHODS
Microorganisms
Combined Activity of Gentamicin with other

Antibiotics
From a total of 460 samples collected from volunteers and patients (over a five months period between
October 2006 and February 2007) after informed
consent and ethical approval, 20 ESBL producers
were identified. From these 8 were recruited into
the antimicrobial combinatorial therapeutic studies. The samples were processed through the following hospitals: respectively from four hospitals
comprising University of Nigeria Teaching Hospital (UNTH); Enugu, National Orthopaedic Hospital, Enugu (NOHE), Ntasiobi Ndinona Afufu
A 1000 g/ml stock solution of gentamicin and

5000 g/ml each of Augmentin (Amoxicillin and
clavulanic acid), cefoxitin, ceftriaxone, cefotaxime,
imipenem and ceftazidime were prepared by dissolving in appropriate quantity of sterile distilled water.
Varying proportion of Gentamicin and the other
antibiotics ranging from 0:10 to 10:0 were mixed
according to the continuous variation checkerboard
method (6). Each proportion of the antibiotics in
20
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TABLE 1. Minimum Inhibitory Concentration for Organisms (MIC) (g/ml)

S/N
1
2
3
4
5
6
7
8
Tag
Cm1
Cm2
Hp3
Hp4
Cm5
Cm6
Hp7
Hp8
Isolates/Source
K. pneumoniae
K. pneumoniae
K. oxytoca
K. pneumoniae
E. coli
K. oxytoca
P. aeruginosa
K. pneumoniae
GENT
12.5
50
25
3.125
1.562
3.125
0.781
1.562
AMC
3.125
25
3.125
6.250
50
3.125
0.781
25
CRO
50
6.25
100
100
1.562
100
6.25
6.25
CIP
100
100
50
50
12.5
100
1.562
100
PEF
100
6.25
100
25
6.25
100
1.562
1.562
CXM
25
1.562
6.25
6.25
12.5
6.25
1.562
1.562
GEN, Gentamicin; AMC, Amoxycillin/Clavulanic acid; CRO, Ceftriaxone; CIP=Ciprofloxacin; PEF, Pefloxacin; CXM, Cefuroxime; Cm,
Community isolates; Hp, Hospital isolates
Interestingly, this bacteria genus is associated with

tremendous secretion and release of the beta-lactamase enzymes further correlating the thrust of this
present research since they were selected (as we
earlier reported in Afunwa et al, 2011) for further
antibiotics combinatorial studies. We then quickly
shifted to develop a simple antibiotics cocktail arrangement to be compatible with global settings
including developing countries where cost of antibiotics treatment are crucial considerations towards
good and effective clinical outcomes.
Figures 1-5 present the outcome of the antibiotic
combinatorial checkerboard assay. Gentamicin-pefloxacin combinations (Figure 1) were largely synergistic against the community isolates in contrast to
hospital isolates except Hp 3 and 4. It is a somewhat
similar scenario for Gentamicin-Amoxicillin/Clavulanic acid cocktail (Figure 2) where again the community isolates were more susceptible synergistically
than the hospital isolates. Thus, the same trend obtains for the Gentamicin-Amoxicillin/Clavulanic
acid case. In the case of Gentamicin-Ceftriaxone
(Figure 3), their combined effect holds no potential
advantage among hospital isolates except in strain
Hp4 where synergism is recorded. There is also no
remarkable utility against community strains where
combined effect simply approached additivity. In a
remarkable trend shift, in Figure 4, Gentamicin-Cefuroxime combination was clearly synergistic against
community isolates. Nevertheless, hospitals isolates
responded differently except isolate Hp4. The most
potent synergism was recorded by Gentamicin-Ciprofloxacin admix (Figure 5) against the plethora of
combination was serially diluted (2 fold) with sterile

water. Thereafter, 1 ml of each of the drug combinations was seeded in a Petri dish together with 19
ml of sterile nutrient agar. It was allowed to stand
for 1 hour to solidify. An aliquot, 0.1ml equivalent
of 0.5 ml MacFarland standard of ESBL producing
organisms was streaked on the surface of the Mueller
Hinton agar plate. The set up was done in triplicate
with a control containing no antibiotics and these
were incubated at 37oC for 24 hours and observed
for growth. The MICs of the various combinations
were determined and interactions between the antimicrobial agents were assessed by determining their
Fractional Inhibitory Concentration [FIC] index using the relationship:
FIC index = FIC A + FIC B
FIC A= MIC of Drug A in combination with Drug B
MIC of Drug A alone
FIC B= MIC of Drug B in combination with Drug A
MIC of Drug B alone
The activity index (AI) =log FIC index
A and B are two antibiotics being combined; MIC,
Minimum Inhibitory Concentration; Drug is antimicrobial agent.
RESULTS
Table 1 shows the result of the antimicrobial susceptibility study and minimum inhibitory concentration (MIC) (g/ml). The MIC values recorded
show rather staggered effects however the Klebsiella
organisms displayed its values for the conventional
cephalosporins and fluroquinolones antibiotics.
21
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FIGURE 1. Susceptibility of ESBL Isolates to Gentamicinpefloxacin antibiotics cocktail

Varying proportions of Gentamicin and Pefloxacin antibiotics
ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller
Hinton agar. The ESBL producing organism was streaked on
the surface of the agar plate. The set up was done in triplicate
with a control containing no antibiotics and these were incubated at 37oC for 24 hours and observed for growth. The MICs
of the various combinations were determined and interactions
between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios
where FIC index value below unity signifies synergism, 1 ;
indifference, and 2; antagonism (Cm1, Cm2, Cm5, and Cm6
represent community isolates, while Hp3, Hp4, Hp7, and Hp8
represent hospital isolates respectively).
FIGURE 2. Susceptibility of ESBL Isolates to GentamicinAmoxicillin/Clavulanic acid antibiotics cocktail

Varying proportions of Gentamicin and Amoxicillin/Clavulanic
acid antibiotics ranging from 1:9 to 9:1 were mixed according
to the continuous variation checkerboard method and seeded
into Mueller Hinton agar. The ESBL producing organism was
streaked on the surface of the agar plate. The set up was done
in triplicate with a control containing no antibiotics and these
were incubated at 37oC for 24 hours and observed for growth.
The MICs of the various combinations were determined and
interactions between the antimicrobial agents were assessed
by determining their Fractional Inhibitory Concentration [FIC]
index ratios where FIC index value below unity signifies synergism, 1 ; indifference, and 2; antagonism (Cm1, Cm2,
Cm5, and Cm6 represent community isolates, while Hp3,
Hp4, Hp7, and Hp8 represent hospital isolates respectively).
the community strains. This was nevertheless only

reproduced in the strains Hp 3 and 4 of the hospital
isolates.
otic combinations represent a therapeutic option in

the treatment of a host of bacterial infections, as a
result of the increasing appearance of multi-resistant
microorganisms. In treatments involving antibiotics like rifampicin, combination therapy is used to
avoid the appearance of antimicrobial resistance
in the infectious agent (9-10). In other treatments,
combinations are used in order to enhance the effect
of individual antimicrobials by means of synergic
interactions. The optimal antibiotic combinations
are commonly obtained from classical suscepti-
DISCUSSION
The wide use of antibiotics in the treatment of bacterial infections has led to the emergence and spread
of resistant strains. Combined antibiotic therapy
may produce synergistic effects in the treatment of
bacterial infection and has been shown to delay the
emergence of antimicrobial resistance (7-8). Antibi22
http://www.jhsci.ba
FIGURE 3. Susceptibility of ESBL Isolates to GentamicinCeftriaxone antibiotics cocktail

Varying proportions of Gentamicin and Ceftriaxone antibiotics
FIGURE 4. Susceptibility of ESBL Isolates to GentamicinCefuroxime antibiotics cocktail

Varying proportions of Gentamicin and Cefuroxime antibiotics
bility tests based on diffusion and dilution (broth

microdilution test or Checkerboard), as described
by the NCCLS. So far, the emergence of ESBL has
compromised the utility of some currently preferred
antibiotics of choice for some bacteria infectious diseases. Unfortunately, the low turnover rate for newer
antibiotics to replace the compromised ones continues to hamper treatment. Therefore, the possibility
of re-inventing effective use protocol through fresh,
seldom applied antibiotics combinatorial platform
may prove a useful alternative approach to overcome
this clinical fisticuffs. There are three potential advantages to using combination therapy: firstly, an
increased likelihood that the infecting pathogen will
be susceptible to at least one of the components of
the regimen; secondly, prevention of emergence of
resistance (11-12) and thirdly, reduced mortality,
perhaps because of an additive or even synergistic
effect of the combination (13-16).
Checkerboard evaluations as a means of monitoring
the combined activities of antimicrobial agents is
based on the general outcome that FIC index value
23
http://www.jhsci.ba
to have relationship to the use practice of the antibiotics in the settings. Gentamicin and pefloxacin are
usually largely abused among the populace within
the communities (17) and as such could readily develop resistance against community bacteria isolates
while maintaining effectiveness against the hospitals
bacteria strains. The reverse trend appears true for
the Gentamicin-Amoxicillin/Clavulanic acid case
scenario. In the case of Gentamicin-Ceftriaxone,
their combined effect holds no potential advantage
among hospital isolates except in strain Cm4 where
synergism is recorded. There is also no remarkable
utility against community strains where combined
effect simply approached additivity. In a remarkable
trend shift, Gentamicin-Cefuroxime combination
was clearly synergistic against community isolates.
Nevertheless, hospitals isolates responded differently except isolate Hp4. The most potent synergism
was recorded by Gentamicin-Ciprofloxacin admix
against the plethora of the community strains. This
was notably also reproduced in the strains Hp 3 and
4 of the hospital isolates, thus making this combination a most favourable cocktail for use against these
ESBL-producing bacteria.
Developing new therapeutic strategies using existing seldom utilized antimicrobial combinations still
present useful option for tackling resistant microbial
circumstances including the menace of the ESBLproducing bacteria (18-20). By simply adjusting the
combinations and amounts of antibiotics applied
could well be the fulcrum to tilt the balance in favour of effective therapy against these categories of
infectious agents. Good antimicrobial monitoring
exercise and corresponding antimicrobial screening
activities should work towards a dynamic approach
to generate effective treatment options using combination therapy. This would then fill a gaping void
created by the ever-widening scourge of persistent
and newer emerging resistant bacteria infections.
FIGURE 5. Susceptibility of ESBL Isolates to GentamicinCiprofloxacin antibiotics cocktail

Varying proportions of Gentamicin and Ciprofloxacin antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller
below unity signifies synergism, 1 ; indifference,

and 2; antagonism. Therefore, using checkerboard
evaluation technique our investigation revealed that
gentamicin-pefloxacin combinations were largely
synergistic against the hospital isolates in contrast to
community isolates except Cm 3 and 4. It is however a somewhat opposite scenario for GentamicinAmoxicillin/Clavulanic acid cocktail where the community isolates were more susceptible synergistically
than the hospital isolates. These observations appear
CONCLUSION
This particular study has shown the importance of

keeping the wheel of progress in a dynamic state for
continued antimicrobial solutions as cheap alternatives expected to find practical usefulness in clinical
settings especially in the tropics.
24
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ACKNOWLEDGEMENTS
64.
7. Aiyegoro OA, Okoh AI. Use of bioactive plant products in combination with
standard antibiotics: implications in antimicrobial chemotherapy. J Med
Plant Res 2009; 3: 1147-1152.
The authors wish to appreciate the technical support of personnel of the Medical Laboratory and
Microbiology Laboratory Units of the following institutions: Division of Pharmaceutical Microbiology
Department of Pharmaceutics, University of Nigeria; National Orthopaedic Hospital Enugu; University of Nigeria Teaching Hospital, Enugu (UNTH);
Nona Afulu Hospital, Enugu.
8. Adwan G, Mhanna M. Synergistic effects of plant extracts and antibiotics

on Staphylococcus aureus strains isolated from clinical specimens. MiddleEast J Sci Res 2008; 3: 134-139.
9. Van der Auwera P, Meunier-Carpentier F, Klastersky J. Clinical study of
combination therapy with oxacillin and rifampicin for staphylococcal infections. Review of Infectious Diseases 5, Suppl. 1983; 3: 515-22.
10. Zeheriya UI, Rabih OD, Glenn CL, Thomas B. Efficacy of antibiotics alone
for orthopaedic device related infections. Clinical Orthopaedics Research
1996; 332: 184-9.
11. Milatovic D, Braveny I. Development of resistance during antibiotic therapy.
Eur J Clin Microbiol 1987; 6: 234-44.
COMPETING INTERESTS
The authors declare that they have no competing

interests.
12. Wade JC. Antibiotic therapy for the febrile granulocytopenic cancer patient:
combination therapy versus monotherapy. Rev Infect Dis 1989; 11 (7):
S1572-81.
13. Giamarellou H. Aminoglycosides plus beta-lactams against gram-negative
organisms. Evaluation of in vitro synergy and chemical interactions. Am J
Med 1986; 80: 126-37.
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et al. High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extended-spectrum beta-lactamases in Dar es Salaam,
Tanzania. J. Clin. Microbiol. 2005; 43: 745-749.
14. Giamarellou H, Zissis NP, Tagari G, Bouzos J. In vitro synergistic activities

of aminoglycosides and new beta-lactams against multiresistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 1984; 25: 534-36.
2. Borer A, Gilad J, Menashe G, Peled N, Riesenberg K, Schlaeffer F. Extended-spectrum beta-lactamase-producing Enterobacteriaceae strains in
community-acquired bacteremia in southern Israel. Med. Sci. Monit. 2002;
8: CR44-47.
15. Klastersky J, Hensgens C, Meunier-Carpentier F. Comparative effectiveness of combinations of amikacin with penicillin G and amikacin with carbenicillin in gram-negative septicemia: doubleblind clinical trial. J Infect Dis
1976; 134: S433-40.
3. Brigante G, Luzzaro F, Perilli M, Lombardi G, Coli A, Rossolini GM, Amicosante G, Toniolo A. Evolution of CTX-M-type -lactamases in isolates of
Escherichia coli infecting hospital and community patients. Int. J. Antimicrob. Agents 2005; 25: 157-162.
16. Klastersky J, Zinner SH. Synergistic combinations of antibiotics in gram

negative bacillary infections. Rev Infect Dis 1982; 4: 294301.
17. Esimone CO, Nworu CS, Udeogaranya OP. Utilization of antimicrobial
agents with and without prescription by outpatients in selected pharmacies
in South- Eastern Nigeria. Pharm Sci Journal. 2007; 11096 p6.
4. Colodner R, Rock W, Chazan B, Keller N, Guy N, Sakran W, Raz R. Risk

factors for the development of extended-spectrum beta-lactamase-producing bacteria in nonhospitalized patients. Eur. J. Clin. Microbiol. Infect. Dis.
2004; 23: 163-167.
18. Albinu I, Odugbemi P, Brian JM. Extended spectrum beta lactamases in

isolates of klebsiella species and Escherichia coli from Lagos, Nigeria. Nig.
Journal of Health and Biomed. Science. 2003; 2 (2): 53-60.
5. Afunwa RA, Odimegwu DC, Iroha RI, Esimone CO. Antimicrobial resistance status and prevalence rates of extended spectrum beta-lactamase
producers isolated from a mixed human population. Bosnian Journal of
Basic Medical Sciences. 2011; 11 (2): 91-96.
19. Fashae K, Albinu I, Ogunsola E, Odugbemi T, Mee JB. Extended Spectrum

Beta-Lactamases in Kebsiella pneumonia isolates from septicaemic children in Ibadan, Nigeria. Nig. J. Health and Biomed. Sc. 2004; 3: (2): 79-84.
20. Iroha IR, Amadi ES, Adikwu MU, Esimone CO. Detection of plasmid borne
extended spectrum beta lactamase enzymes in clinical isolates of Escherichia coli from a community general hospital. Int. J. Mol. Adv. Sc. 2009: 4
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6. Okore VC. Evaluation of chemical antimicrobial agents. In: Okore VC. (ed).
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25
Amila Kapetanovi, Dijana Avdi Journal of Health Sciences 2014;4(1):26-30
http://www.jhsci.ba

RESEARCH ARTICLE
Open Access
Influence of cigarette smoking on bone mineral

density in postmenopausal women with estrogen
deficiency in menstrual history
Amila Kapetanovi1, Dijana Avdi2,3
1
Medical Rehabilitation Center Fojnica, Fojnica, Bosnia and Herzegovina. 2Faculty of Health Studies, University of Sarajevo,
Bolnika 25, Sarajevo, Bosnia and Herzegovina. 3Clinic for orthopedics and traumatology, Clinical Center of the University
of Sarajevo, Sarajevo, Bosnia and Herzegovina.
ABSTRACT
Introduction: Estrogen deficiency leads to bone mass loss and increased risk for osteoporosis. The aim
of this study was to examine influence of cigarette smoking on bone mineral density in postmenopausal
women with estrogen deficiency in menstrual history.
Methods: The total of 100 postmenopausal women living in Sarajevo area, aged 50-65 years, with estrogen deficiency in menstrual history participated in this prospective study. The subjects were divided in
two groups, examination and control group, based on bone mineral density values. The women in the
examination group had osteoporosis while in the control group were women with osteopenia or normal
bone mineral density. Bone mineral density was measured at the lumbar spine and proximal femur by
DualEnergy Xray Absorptiometry using Hologic QDR-4000 scanner. Smoking habits were assessed for
each subject.
Results: The average number of cigarettes smoked per day in women with estrogen deficiency in menstrual history was 14.86 in the examination group and 4.67 in the control group. The difference in the
average number of cigarettes smoked per day between the two groups was statistically significant (p
<0.01). The coefficient of linear correlation between T score and the number of cigarettes smoked per day
among women with estrogen deficiency in menstrual history in the examination group was statistically
significant (p<0.01). The coefficient of linear correlation between T score and the number of cigarettes
smoked per day among women with estrogen deficiency in menstrual history in the control group was
statistically significant ( p<0.05).
Conclusion: Results of this study suggest that cigarette smoking has negative impact on bone mineral
density and that healthy lifestyle (no smoking) has the potential to reduce bone loss in postmenopausal
women with estrogen deficiency in menstrual history.
Keywords: osteoporosis, cigarette smoking
*Corresponding author: Amila Kapetanovi

Medical Rehabilitation Center Fojnica,
Fojnica, Bosnia and Herzegovina
E-mail: nermin1a@bih.net.ba
INTRODUCTION
Osteoporosis is a skeletal disorder characterized by

compromised bone strength (bone strength primarily reflects the integration of bone density; bone
Submitted March 5, 2014/Accepted April 12, 2014
2014 Amila Kapetanovi, Dijana Avdi; licensee University of Sarajevo - Faculty of Health
Studies. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
http://www.jhsci.ba
METHODS
quality refers to architecture, metabolic turnover,

damage accumulation and demineralization) predisposing a person to an increased risk of fracture (1).
The pathogenesis of osteoporosis include: failure to
achieve a skeleton of optimal strength during growth
and development, excessive bone resorption resulting in loss of bone mass and disruption of architecture and failure to replace lost bone due to defects
in bone formation (2). Sex steroids (estrogens and
androgens) slow the rate of bone remodeling and
protect against bone loss acting on osteoclastogenesis and osteoblastogenesis, osteoblast and osteocyte
apoptosis (3). Estrogen deficiency increases the rate
of remodeling and increases imbalance in the bone
multicellular unit resulting in bone loss and structural decay after menopause (4). A premature menopause, particularly when surgically induced before
age 45 years, is strong determinant of bone density
and increased risk of fracture (5). Late menarche is
also associated with low bone mineral density (6,7).
The later the menarche and the earlier the menopause, the higher the degree of osteoporosis (7).
Cigarette smoking affects the bone duo direct toxic
effects on osteoblasts/osteoclasts activity of nicotine,
and indirect actions on sex and adrenocortical hormones, vitamin D, intestinal calcium absorption,
vessels and oxygen supply (8). Smoking affects pituitary, thyroid, adrenal, testicular and ovarian function, calcium metabolism and the action of insulin
and the major salient clinical effects are the increased
risk and severity of Graves hyperthyroidism and opthalmopathy, osteoporosis and reduced fertility (9).
Some normal, estrogen-dependent physiologic processes are affected by smoking, making osteoporosis
and premature menopause more common among
women who smoke (10). Smoking reduces the production of estrogen by the ovaries, causing changes
in hepatic estrogen metabolism, showed toxic effects
on ovarian follicles and the bone cells, reduces the
conversion of androgen into estrogen, the concentration of sex hormone binding globulin (SHBG),
which binds circulation estrogens, is higher in women who smoke, and lower concentrations of the biologically active estrogens (9, 11, 12). The aim of this
study was to examine influence of cigarette smoking
on bone mineral density in postmenopausal women
with estrogen deficiency in menstrual history.
The total of 100 postmenopausal women living in

Sarajevo area, aged 50-65 years, with estrogen deficiency in menstrual history participated in this
prospective study. The subjects were divided in two
groups, examination and control group, based on
bone mineral density values. The women in the
examination group (n=50) had osteoporosis. The
women in the control group (n=50) had osteopenia
or normal bone mineral density. Bone mineral density was measured at the lumbar spine and proximal
femur by DualEnergy Xray Absorptiometry using
Hologic QDR-4000 scanner. Smoking habits were
assessed for each subject. The inclusion criteria were:
postmenopausal women with estrogen deficiency
in menstrual history (fewer than 30 years of menstruation, menopause before age 45 years), women
aged 50-65 years, women who live in the Sarajevo
area, women who do not use hormone replacement
therapy, women whose finding's of bone densitometry was at the level of osteoporosis, women whose
finding's of bone densitometry was at the level of
osteopenia or normal. The exclusion criteria were:
postmenopausal women without estrogen deficiency in menstrual history, women younger than
50 and older than 65 years, women who do not
live in the Sarajevo area, women who are not postmenopausal, women who use hormone replacement
therapy, women who have a disease that can cause
osteoporosis, women who use drugs that may cause
osteoporosis.
The results were statistically analyzed. Statistical significance between examination and control group in
cigarette smoking was tested by Student's t-test. The
coefficient of linear correlation between cigarette
smoking and bone mineral density was calculated. P
< 0.05 was considered statistically significant.
RESULTS
The average age of women with estrogen deficiency

in their menstrual history in the examination group
was 58.48 years, and in the control group was 57.30
years. There was no statistically significant differences between these two groups, t = 1.169.
27
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The coefficient of linear correlation between T scores

and the number of cigarettes smoked per day among
women with estrogen deficiency in menstrual history in the examination group was statistically significant, r = - 0.671; p < 0.01. The coefficient of
linear correlation between T scores and the number
of cigarettes smoked per day among women with
estrogen deficiency in menstrual history in the control group was statistically significant, r = - 0.350;
p < 0.05.
TABLE 1. The coefficient of linear correlation between T
scores and the number of cigarettes smoked per day among
women with estrogen deficiency in menstrual history
FIGURE 1. The average age of women with estrogen deficiency in menstrual history. t = 1,169; no statistically significant
Parameters
The coefficient of
linear correlation
The average number of cigarettes smoked per day in

women with estrogen deficiency in menstrual history stood at 14.86 in the examination group and in
the control group 4.67. The difference in the average number of cigarettes smoked per day between
the two groups was statistically significant, t = 6.009,
p < 0.01.
Examination
group A
r = - 0.671
p < 0.01
Control
group A
r = - 0.350
p < 0.05
DISCUSSION
Estrogen deficiency leads to bone mass loss and

increased risk for osteoporosis. Late menarche and
early menopause are associated with bone loss. The
objective of the study of Parker et al. was to investigate the association between age at menarche, age
at menopause, and years of menstruation with incidence of osteoporosis and assess the impact of prenatal exposure to diethylstilbestrol (DES), a synthetic
estrogen, on such associations. The results support
the hypothesis that lifetime cumulative exposure to
estrogens is protective against osteoporosis (6). The
results of the study of Li et al. showed significant
increase in osteoporosis of lumbar spine in women
with the age of menarche > or = 17 compared with
women with age of menarche < or = 13. Among all
women aged between 55 65, there was significant
increase in osteoporosis in women with the menopause age < or = 48, compared with the menopause
age > or = 54 (7). In our previous study we evaluated
the influence of menstrual factors (years between
menarche and menopause, years since menopause)
on bone mass loss in Bosnian postmenopausal women. Average years between menarche and menopause in women with osteoporosis was significant
lower than in women in control group (osteopenia or normal mineral bone density values). Average years since menopause was significant higher
FIGURE 2. The average number of cigarettes smoked per

day among women with estrogen deficiency in menstrual history (p < 0.01)
28
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in women with osteoporosis than in women with

osteopenia or normal bone mineral density values
(13). Meta-analyses on the effects of smoking on
the bone revealed that current smokers sustained
decreased bone mass and increased fracture risk at
age 50 years and older (14). Numerous studies point
to the negative effects of smoking on bone tissue.
Giampietro et al. assessed the relative impact of cigarette smoking, statin use, genetic polymorphisms,
and one-way interaction of these factors on development of osteoporosis in postmenopausal women.
The results suggested a role for genetic variation in
the interleukin 6 (IL6) and the lipoprotein receptorrelated protein 5 (LRP5) in conferring risk for osteoporosis in Caucasian women, with the latter manifest only in smokers (15). Ward and Klesges studied
cross-sectional and prospective human studies that
provided a quantitative measure of bone mass (Xray, absorptiometry, or computed tomography) as
a function of cigarette smoking exposure. Smokers
had significantly reduced bone mass compared with
nonsmokers (never and former smokers) at all bone
sites. Overall, effects were greatest in men and in
the elderly, and were dose-dependent. In prospective studies, smokers had greater rates of bone loss
over time compared with nonsmokers. Absolute effect sizes at most bone sites were greatest for current
smokers compared with never smokers, intermediate
for current smokers compared with former smokers,
and lowest for former smokers compared with never
smokers, suggesting that smoking cessation may
have a positive influence on bone mass (16). Brook
et al. assessed how different trajectories of women's
smoking, covering the ages 40 to 48 years, relate to
osteoporosis at age 65. The chronic/heavy smokers
were significantly more likely than the non-smokers
to report having osteoporosis. Quitters and moderate smokers did not differ significantly from nonsmokers on the osteoporosis measure (17). The association between secondhand smoke exposure and
lumbar and femoral neck osteoporosis was assessed
in postmenopausal never-smoking Korean women
in the study of Kim et al. The finding of the study
was that in postmenopausal never-smoking Korean
women, exposure to secondhand smoke was positively associated with osteoporosis (18). Jenkins and
Denison found that former and current smoking
increased the risk of hip fracture in population of
postmenopausal women living in rural and urban

areas of Northwest Texas (19). In the study of Cummins et al. showed that although hereditary factors
strongly contribute to bone health, behavioural factors can modulate the genetically determined pattern of skeletal modelling and remodelling. Smoking
was the strongest behavioural predictor of lumbar
and femoral bone mineral density in pre- and postmenopausal adult Irish women (20). The aim of the
present study was to examine influence of cigarette
smoking on bone mineral density in postmenopausal women, aged 50-65 years living in Sarajevo area
with estrogen deficiency in their menstrual history.
The results showed that smoking has a negative effect
on bone mineral density in postmenopausal women,
aged 50-65 years living in Sarajevo area with estrogen deficiency in their menstrual history. The average number of cigarettes smoked per day in women
with estrogen deficiency in menstrual history stood
at 14.86 in the group of women with osteoporosis
and in the group of women without osteoporosis
4.67. The difference in the average number of cigarettes smoked per day between the two groups was
statistically significant (p < 0.01). The coefficient of
linear correlation between T scores and the number
of cigarettes smoked per day among women with
estrogen deficiency in menstrual history was statistically significant in the group of women with osteoporosis (p < 0.01) and in the group of women without osteoporosis (p < 0.05). It was shown that the
negative effects of smoking on bone mineral density
are dose-dependent. The deficit of estrogen is associated with a decrease in bone mass, but healthy
lifestyle (no smoking) has the potential to preserve
bone mass in postmenopausal women with estrogen
deficiency in their menstrual history. Encouragement of lifestyle alterations, including smoking cessation, should be a major component of any bone
therapeutic programme (21).
CONCLUSION
The results of this study suggest that cigarette smoking has influence on bone mineral density and increases bone loss in postmenopausal women, aged
50-65 years living in Sarajevo area, with estrogen deficiency in their menstrual history. Further healthy
lifestyle (no smoking) has positive impact on bone
29
http://www.jhsci.ba
in postmenopausal women with estrogen deficiency

in their menstrual history.
10. Spangler JG. Smoking and hormone-related disorders. Primary Care 1999;
26: 499511.
CONFLICT OF INTEREST
12. Bartl R. Osteoporose: Pravention Diagnostik Therapie. Georg Thieme

Verlag KG, 2004.
The authors declare no conflict of interest.
13. Kapetanovi A, Avdi D, Markovi K. The influence of menstrual factors on

bone mass loss in bosnian postmenopausal women. HealthMed, 2007;
1(2): 2-9
REFERENCES
14. Iki M. Osteoporosis and smoking. Clin Calcium. 2005 Jul;15(7):156-8.
1. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy. Osteoporosis prevention, diagnosis and therapy. JAMA,
2001;285:785-795
2. Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J. Clin. Invest. 2005;115:3318-3325.
15. Giampietro PF, McCarty C, Mukesh B, McKiernan F, Wilson D, Shuldiner A,

Liu J, LeVasseur J, Ivacic L, Kitchner T, Ghebranious N. The role of cigarette smoking and statins in the development of postmenopausal osteoporosis: a pilot study utilizing the Marshfield Clinic Personalized Medicine
Cohort. Osteoporos Int. 2010 Mar;21(3):467-77.
3. Manolagas SC, Kousteni S, Jilka RL. Sex Steroids and Bone. Recent Prog
Horm Res. 2002;57:385-409.
16. Ward, KD, Klesges RC. A meta-analysis of the effects of cigarette smoking
on bone mineral density. Calcif Tissue Int. 2001 May;68(5): 259-270.
4. Seeman E, Delmas PD. Bone quality: the material and structural basis of
bone strength and fragility. N. Engl J Med 2006;354(21):2250-2261.
17. Brook JS, Balka EB, Zhang C. The smoking patterns of women in their
forties: their relationship to later osteoporosis. Psychol Rep. 2012
Apr;110(2):351-62.
11. U.S. Department of Health and Human Services. Bone Health and Osteoporosis: A Report of the Surgeon General. Rockville, MD; 2004.
5. World Health organization. Prevention and management of osteoporosis.

WHO Technical Report Series 921, 2003.
18. Kim KH, Lee CM, Park SM, Cho B, Chang Y, Park SG, Lee K. Secondhand
smoke exposure and osteoporosis in never-smoking postmenopausal
women: the Fourth Korea National Health and Nutrition Examination Survey. Osteoporos Int. 2013 Feb;24(2):523-32.
6. Parker SE, Troisi R, Wise LA, Palmer JR, Titus-Ernstoff L, Strohsnitter

WC, Hatch EE. Menarche, menopause, years of menstruation, and the
incidence of osteoporosis: the influence of prenatal exposure to diethylstilbestrol. J Clin Endocrinol Metab. 2014 Feb;99(2):594-601.
19. Jenkins MR, Denison AV. Smoking status as a predictor of hip fracture risk
in postmenopausal women of Northwest Texas. Prev Chronic Dis. 2008
Jan;5(1):A09.
7. Li HL, Zhu HM. Relationship between the age of menarche, menopause

and other factors and postmenopause. Osteoporosis Zhonghua Fu Chan
Ke Za Zhi. 2005 Dec;40(12):796-8
20. Cummins NM, Jakeman PM, Sestak I, Murphy N, Carroll P. The effect of
behavioural risk factors on osteoporosis in Irish women. Ir J Med Sci. 2013
Mar;182(1):97-105.
8. Abate M, Vanni D, Pantalone A, Salini V. Cigarette smoking and musculoskeletal disorders. Muscles Ligaments Tendons J. 2013 Jul 9;3(2):63-9.
21. Wong PK, Christie JJ, Wark JD. The effects of smoking on bone health.
Clin Sci (Lond). 2007 Sep;113(5):233-41.
9. Kapoor D, Jones TH. Smoking and hormones in health and endocrine disorders. European Journal of Endocrinology, 2005;152(4):491-499.
30
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Sabina Alispahi et al. Journal of Health Sciences 2014;4(1):31-35

RESEARCH ARTICLE
Open Access
Characteristics of Patients Involved in

Psychotherapy in Bosnia and Herzegovina
Sabina Alispahi1*, Enedina Hasanbegovi-Ani1, enita Tuce1,
Nina Hadiahmetovi1, Aneta Sandi2
1
Faculty of Philosophy, University of Sarajevo, Psychology Department. Franje Rakog 1, 71 000 Sarajevo, Bosnia and
Herzegovina. 2Private psychiatric practice with the cabinet for psychotherapy Dr. Sandi, Grbavika 58, 71000 Sarajevo,
Bosnia and Herzegovina.
ABSTRACT
Introduction: The aim of this study was to determine the demographic and clinical characteristics of Bosnian and Herzegovinian patients involved in psychotherapeutic treatments in order to explore the current
situation of psychotherapy in Bosnia and Herzegovina.
Methods: The study included 213 patients (154 women and 47 men) undergoing diverse psychotherapeutic treatments. Data about demographic and clinical characteristics were collected by questionnaire.
Following characteristics were documented: age, sex, education, employment status, marital status, specific problem that got the client involved in psychotherapy, type of psychotherapy, and use of psychopharmacology.
Results: Majority of the patients undergoing psychotherapy are age up to 40 and female. They are by
vast majority holding a university degree and are employed. Nearly equal number of patients is living in
partnership or marriage compared to single or never been married. Most frequent reasons for getting
involved in the psychotherapy treatment are of the intrapersonal nature (depression, anxiety and panic
attacks). Majority of the patients were involved in gestalt and cognitive behavioral psychotherapy, and at
the same time majority of those were not prescribed medicaments.
Conclusions: We point out and overview some of the most prominent socio-demographic traits of patients undergoing psychotherapy, the ones that could be important in the future research with the higher
degree of control. In the terms of personal initiative, psychotherapy stops being a taboo in Bosnia and
Herzegovina. However, there is still a long path until it reaches integration in daily life of the people.
Keywords: psychotherapy, Bosnia and Herzegovina.
INTRODUCTION
*Corresponding author: Sabina Alispahi
Faculty of Philosophy, University of Sarajevo, Psychology Department. Franje Rakog 1, 71 000 Sarajevo, Bosnia and Herzegovina
Phone:+ 387 33 253 174
E-mail: sabina_alispahic@hotmail.com
Submitted February 12, 2014/ Accepted April 20, 2014
Due to the evident increase of number of patients

undergoing different psychotherapy treatments, various research were conducted in the past years questioning the factors associated with the application
of psychotherapy and improvement of the status of
2014 Sabina Alispahi et al.; licensee University of Sarajevo - Faculty of Health Studies. This
http://www.jhsci.ba
patients with regards to the various emotional and

cognitive disorders (1-8). Importance of exploring
the trend of using the psychotherapy also lies in the
fact that this describes the modalities of use of the
medical insurance of the citizens. A recent study explored demographic, sex, ethnic and cultural traits
of psychotherapy patients and found that women
and white race in general are more frequent users of
psychotherapy (9-11). For this reason the cultural
context within which psychotherapeutic treatments
were conducted came out as significant. In Europe
and USA the patients are more prone to undergo
psychotherapy than psychopharmacological treatment and it is also registered that information and
options about the psychotherapeutic treatments are
quite available.
Until recent times, in non Western societies psychological difficulties were treated by the means of traditional cultural customs. However, increased urbanization and globalization of non Western societies
led towards development of the interest for Western
psychotherapies and their practical application (12).
This increased the consciousness about the benefits
of psychotherapy and its outcomes (13). However,
there is still a lack of data with regards to the development and application of psychotherapy in non
Western societies (14). According to some indicators,
socio-cultural forms over generations had significant impact on the attitudes about the protection
of mental health and the therapeutic and economic
worth of psychotherapeutic services.
Although it has a long history psychotherapy in Bosnia and Herzegovina (BIH) is still not sufficiently
developed. It does not have a defined status which
indeed belongs to it due to the efficacy as well as
the width of psychopathology it addresses. During
the last couple of years education in different psychotherapeutic modalities in BIH begun (gestalt,
cognitive-behavioral, reality oriented, family psychotherapy, transactional therapy, psychodynamic
psychotherapy and psychodrama). This is the reason
why there are more and more licensed psychotherapists in the country. Although it is in the stage of development, currently there is a lack of research projects exploring the status of psychotherapy in BIH, as
well as the issues addressing the patients.
The aim of this research was to explore demographic
and clinical traits of patients undergoing psychotherapy (different therapeutic approaches) and by
those means gather data about the actual status of
psychotherapy in BIH.
METHODS
Participants
The study included 213 patients (154 women and

47 men), with mean age M = 34 (SD = 16.1). Participants were patients involved in different psychotherapies (gestalt, cognitive-behavioural, systemicfamily and psychodynamic therapy), who were
included in the psychotherapeutic treatment, regardless of the type of their problem. Data were collected from patients in five cities in BIH: Sarajevo,
Mostar, Prijedor, Banja Luka and Tuzla. The study
involved patients who meet the following criteria:
older than 18 years, currently not psychotic, and
dont have recorded cognitive impairments and associated intellectual disability.
Instruments
Questionnaire of demographic and clinical traits was

designed and included the following traits of the patients: age, sex, place of residence, education level,
employment status, marriage status, financial status,
type of the problem that led the client towards psychotherapeutic treatment, psychotherapeutic modality, and weather pharmacotherapy was included
in the treatment or not.
Procedure
Data were collected by individual work method. The

authors of the study first contacted psychotherapists
from several cities in BIH, who asked their current
patients to complete a Demographic and clinical client characteristics questionnaire, which required an
average of 5 minutes of time.
Data collection was carried out in the central phase
of psychotherapy. All participants got general instruction in which authors explained the goal of research. Participants completed the questionnaire in
the waiting room after psychotherapy sessions, not
to interrupt the time of therapeutic work. The completed questionnaires were returned back to therapist in sealed envelopes. Participation in the survey
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TABLE 1. Demographic traits of patients involved in psychotherapy in Bosnia and Herzegovina
was anonymous and voluntary. All phases of the research process were carried out in accordance with
the ethical principles of scientific research.
Variables
Sex
Males
Females
Age
18-29
30-39
40-49
50-59
60-69
Education level
elementary
high school
higher expertise
university degree
Employment status
Employed
Not employed
Retired
Marriage status
Married/living with partner
Never married
Divorced
Widow
RESULTS
Majority of patients involved in psychotherapy are

women (75%). Also, the majority of our sample
consists of patients of age range between 18 and 40
(73%) (Table 1).
Majority of the patients undergoing psychotherapy
hold a university degree (53%) and are employed
(61%). Speaking of their marriage status it is interesting to note that there is approximately similar
number of those that are in marriage or partnership
relations (42%) and the ones that were never married (41%) (Table 1).
Patients got involved in psychotherapy for a variety
of reasons. Majority of patients, however, suffered
from depression, anxiety and panic attacks (43%).
Interpersonal problems (family hardships, difficulties in marriage, with other people and problems at
working place) were ranked second frequent reason
(31%) for undergoing this kind of treatment. Certain number of patients got involved in psychotherapy because of other various difficulties we categorized in a different group (other), such as various
loses, substance abuse, mobbing, different type of
phobias, personality disorders, etc (Table 2).
Majority of the sample consists of the patients involved in gestalt and cognitive-behavioural psychotherapy (73%) and majority of them were not using
any medications (68%) (Table 2).
% (N=213)
25
75
37
36
19
7
1
0.5
41
6
52.5
61
36
3
43
41
12
4
TABLE 2. Clinical traits of patients involved in psychotherapy

in Bosnia and Herzegovina
Reason for undergoing psychotherapy
Depression
Anxiety
Marriage problems
Panic attacks
Hardships with other people
Family problems
Problems at working place
Other
Psychotherapy type
Gestalt
Cognitive-behavioural
Systemic family psychotherapy
Psychodynamic
Use of medicaments
YES
NO
DISCUSSION
According to the results of the research most frequent patients involved in psychotherapy in BIH
are adults (18 to 40 years old). This data is not a
surprise if we consider the fact that young adults in
todays society face issues and challenges that did
not exist, or were unacknowledged, in previous generations. In recent decades, important demographic,
social and cultural changes have affected the lives
and needs of young adults in many countries around
the world.
Choices for both genders are more numerous for
young adults than they were several decades ago. Ex33
% (N=213)
20
13
13
10
8
7
3
26
37
36
13
7
32
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pectations are less clear about what a person's next

step should be after completing elementary school
education (whether it be high school, college, or
graduate school). In most countries, young adults
are now taking longer time to finish their education, assume full time employment and undertake
personal financial responsibility. At the same time,
young adults are coping with marked levels of mental health issues (14).
Kessler et al. (15) summarized the World Health Organizations (WHO) World Mental Health Survey
data on the incidence and prevalence of major mental health disorders (anxiety, mood, impulse control
and substance use) across 17 countries. Examined
by age group (18 to 34, 35 to 49, 50 to 64, 65+),
the WHO data showed high rates of multiple types
of mental disorders for 18 to 34 year olds, indicating significant levels of distress for this age group in
many countries around the world.
Vast majority of patients in psychotherapy are females, the result of the research in accordance with
results of other researches (6, 7). This could signify
that women are more prone to search psychotherapeutic help. Additional explanation is that women
are exposed to a higher number of socio cultural
stressors (such as problems is marriage, family and
financial difficulties) when compared with men,
and that are for this reason more vulnerable and
susceptible to emergence of psychological difficulties. According to the results of recent research (16),
women, in general, are more susceptible to explore
their emotional as well as to speak of their problems
while undergoing psychotherapy. The mentioned
research confirmed that women use more metaphor
in speech, identify childhood trauma as reasons for
their hardships, and also more frequently verbalize
their anger when compared to man. It is also possible that specific actual cultural context in BIH and
male identity (characterized by strength, dominance,
authority) makes men less prone to search for psychological help.
According to our results patients involved in psychotherapy are by majority holding a university
degree. The results are not consistent when addressing educational status of patients in psychotherapy. According to some research (6, 7, 17) persons
with lower educational level are more rare users of
psychotherapeutic services when compared with

persons with a higher level of education. However,
results of research of more curent date point that
users of pschotherapy are not only more educated
and more successful patients (18). According to
previous research it could be concluded that, most
probably, subjects with higher educational level possess a more developed consciousness of importance
and the usefulness of psychotherapy. Together with
higher economic status this makes the psychotherapeutic treatment more available to them.
Our research brought up the result about the majority of patients being employed. However, a significant number of patients involved in psychotherapy
are also unemployed. Those results are in accordance
with majority of research studies (6, 7). Research
studies did not identify a consistent link among the
use of psychotherapy, employment status and financial income (6, 7).
According to results of our research the most frequent reasons for getting involved in psychotherapy
are of intrapersonal nature (depression, anxiety and
panic attacks) what matches the prevalence of the
mentioned disorders in general population. The
prevalence of panic disorder across the life span, as
noted in epidemiological studies, is between 1.5 and
3%. For the generalized anxiety it amounts to approximately 5%. Majority of anxiety disorders appears in comorbidity with other psychological disorders depression being common by far (19).
Results of the research show that majority of patients got involved in gestalt and cognitive behavioral psychotherapy, as well as that most of them do not
use medication. One of the possible explanations of
those findings is the fact that the noted psychotherapeutic modalities are the most available in the country (according to data available to us the majority of
psychotherapists in BIH are of gestalt and cognitive
behavioral orientation).
Addressing the use of medication it is known that
patients involved in psychotherapy may and may
not use it. There are mostly two combinations that
exist: first, when one expert prescribes medicaments
and at the same time conducts psychotherapy, and
the second where client sees two persons, and each
of the experts leads his own part of the job.
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Acknowledgements
The research is marked by at least two limitations.

The first one relates to the sample: patients are inhabitants of several cities in BIH what makes the
sample not possible to be generalized to wide, general population. Future research should involve
participants from smaller social habitats. Second
limitation is that psychotherapists who took part in
the research come from the private practice. For this
reason future research should include patients from
the public spectrum (e.g. medical health centers).
It would be also interesting to explore whether the
distribution of reasons for searching psychotherapy
matches the general epidemiologic frame of psychological disorders in BIH.
This research could be used as a foundation for future research studies about the users of
psychotherapeutic services in BIH. It is important
that experts from the field of mental health explore
the need for psychotherapeutic services in the country in which concepts, methods and outcomes of
various psychotherapeutic modalities are becoming
more and more appreciated. It is obvious that in the
terms of ones own initiative psychotherapy ceases to
be a taboo. Yet, there is still a long path until it gets
integrated in the daily lives of the citizens.
We thank all psychotherapists included in this research, as well as their patients.

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19. trkalj Ivezi S, Folnegovi malc V, Mimica N. Dijagnosticiranje
anksioznih poremeaja. MEDIX 2007, 71: 56-58.
35
Jozo ori et al. Journal of Health Sciences 2014;4(1):36-39
http://www.jhsci.ba

RESEARCH ARTICLE
Open Access
Evaluation of tumor marker HE4 assay on the

Elecsys 2010 analyzer
Jozo ori, Lejla Hasanbegovi, Aleksandar Bodulovi, Jasminka Muji
Department of Clinical Chemistry, Clinical Center of Sarajevo University, Bolnika 25, 71000 Sarajevo, Bosnia and Herzegovina
ABSTRACT
Introduction: Whey-acidic protein human epididymis protein 4 (HE4) is a new promising biomarker for
epithelial ovarian cancer. The measured HE4 values may depend on the testing procedure used. The aim
of this study was to evaluate the
Methods: We evaluated a HE4 method on Elecsys 2010 analyzer. The method for quantitative determination of HE4 is direct, competitive chemiluminescent immunoassay. For quality control we use Elecsys
PreciControl HE4 1 and 2. HE4 was measure on sera obtained from 56 women ( 20 healthy and 36 with
epithelial ovarian cancer).
Results: The Roche HE4 assays showed a good linearity (r=0.99) and precision (intrassayed total CV<5%).
The median HE4 serum concentrations was significantly higher among EOC patients than healthy females
(p<0,05). Elevated levels HE4 were found in 78 % patients with epithelial ovarian cancer.
Conclusions: The presented results of the analytical evaluation methods for the determination of HE4 on
the Elecsys 2010 analyzer showed an acceptable accuracy and precision.
Keywords: Human epididymis protein 4 (HE4), epithelial ovarian cancer, biomarkers
INTRODUCTION
Epithelial ovarian cancer (EOC) is the fourth most

common cause of cancer mortality in women and
the leading cause of death from gynecological malignancies. The majority of women have advancedstage disease at initial diagnosis and a 5-year survival
of 10-30 %. The median survival after recurrence is
only 2 years despite the advances in chemotherapy
*Corresponding author: Jozo ori
Department of Clinical Chemistry,
Clinical Center of Sarajevo University,
Bolnika 25, 71000 Sarajevo,
Bosnia and Herzegovina
Submitted February 10, 2014/Accepted April 1, 2014
and secondary debulking surgery in selected patients (1).

The human epididymis protein 4 (HE4, also known
as WFDC2) belongs to the family of whey acidic
four-disulfide core (WFDC) proteins with suspected trypsin inhibitor properties. The corresponding
gene codes for a 13 kD protein. In its mature glycosylated form the protein has a molecular weight
of approximately 20-25 kD and consists of a single
peptide chain containing two WFDC domains (2).
HE4 was first determined in the epithelium of the
distal epididymis. It showed low expression in epithelia of respiratory and reproductive tissues including ovary, but high expression in ovarian cancer tis-
2014 Jozo ori et al.; licensee University of Sarajevo - Faculty of Health Studies. This is an
http://www.jhsci.ba
reaction is measured as relative light units (RLUs).

A direct relationship exists between the amount of
HE4 antigen in the sample and RLUs.
For quality control ElecsysPreciControl HE4 1 and
2 (Roche Diagnostics, Germany) were used.
The data were analyzed in SPSS version 18 (Chicago,
IL, USA). Descriptive statistics and Pearson correlation were used. The difference between groups were
considered significant if p<0.05.
sue. High secreted levels can also be found in the

serum of ovarian cancer patients (3).
The measured HE4 value of a patients sample can
vary depending on the testing procedure used. The
laboratory finding must therefore always contain a
statement on the HE4 assay method used. HE4 values determined on patient samples by different testing procedures cannot be directly compared with
one another and could be the cause of erroneous
medical interpretations (4,5).
The assay is to be used as aid in monitoring recurrence or progressive disease in patients with epithelial ovarian cancer. Serial testing for patient HE4 assay values should be used in conjunction with other
clinical methods used for monitoring ovarian cancer
(6).
Moore and colleagues (7) have reported that the
serum biomarker algorithm ROMA (Risk of Ovarian Malignancy Algorithm), which utilizes the dual
marker combination of HE4 and CA125, along
with menopause status, shows good performance for
risk prediction of ovarian malignances (7).
RESULTS
The results of the HE4 assay precision within-run

and between-run analyses are showed in Table 1.
The coefficients of variation (CV%) values for the
within-run precision were 6.9 8.2 %, with those
for between-run ranging 7.9 9.8%. The results
variation was greater at lower concentrations.
The HE4 assay accuracy results are presented in
Table 2.
The statistically significant correlation between
labeled and measured HE4 values was obtained
(r=0.99), presented by the following equation: y=
METHODS
Blood samples were collected from 36 female pa- -1.20+0.99x, where y represents the measured HE4
tients with epithelial ovarian cancer (mean age: 56 levels, and x labeled HE4 levels. An intercept (-1.20)
years, range 48-69 years). Control samples (N=20) presents the systematic errors of the method, which
was not statistically significant (p>0.05).
were collected from healthy females. The blood was
collected into the non-heparinized tubes and kept
30 min at room temperature, then centrifuged for TABLE 1. Within-run and between-run precision of HE4 as15 min at 3000 rpm. The sera were then stored at say
-20 oC until use.
Sample
Sample Mean value
Sd
CV(%)
The serum levels of HE4 were determined using a
value (n) (pmol/L) (pmol/L)
chemiluminescent enzyme immunoassay on the
Within-run
Elecsys 2010 Analyzer (Roche Diagnostics, Germa- PreciControl HE4 1
20
45.5
3.7
8.2
ny) according to manufacturer, s instructions. The
PreciControl HE4 2
20
341.5
23.5
6.9
HE4 assay is a two-step immunoassay for quantita- Between-run
tive determination of HE4 antigen in human serum
PreciControl HE4 1
20
42.4
4.1
9.8
using CMIA technology. In the first step, sample
PreciControl HE4 2
20
347.7
29.2
7.9
and 2H5 anti-HE4 coated paramagnetic micropaticles are combined. HE4 antigen present in the
sample binds to the anti-HE4 coated microparticles. TABLE 2. Accuracy of HE4 assay on Elecsys 2010 analyzer.
After washing, 3D8 anti-HE4 acridinium-lebeled
Calibrators (pmol/L) 20.0 50.0 100.0 500.0 1000.0
conjugate is added to create a reaction mixture
Measured values*
in the second step. Following another wash cycle,
29.7 49.2 98.4 497.1
992.1
(pmol/L)
pre-trigger and trigger solutions are added to the
reaction mixture. The resulting chemiluminiescent *Mean of two measurements of calibrators
37
http://www.jhsci.ba
TABLE 3. HE4 measurements

Group
EOC
Healthy controls
N
36
20
Median
(pmol/L)
139.6
32.1
from 20 to 1000 pmol/L, which is acceptable for

routine clinical use. The CV for precision in this assay is not greater than 10% at the lowest measurable
concentration. The obtained CV% values for precision were for the within-run precision were 6.9 8.2
%, with those for between-run ranging 7.9 9.8%.
The results variation was greater at lower concentrations.
The median concentrations in women test group
were 139.6 (range 34,2 1383,0 ) pmol/L. Reference interval for HE4 ranges between 0 to 74. The
median serum HE4 concentrations were significantly higher among all ovarian cancer patients compared with group of healthy subjects. Elevated levels
HE4 were found in 78 % patients with epithelial
ovarian cancer.
Range
(pmol/L)
34.2 1383.0
11.2 72.3
EOC, epithelial ovarian cancer
Serum samples were collected from 36 ovarian cancer patients (EOC). Results of HE4 measurements
in different groups are shown in Table 3.
The median serum HE4 concentrations were significantly higher among all ovarian cancer patients
compared with group of healthy subjects.
Patients with ovarian carcinoma showed significantly higher HE4 levels of 139.6 (range: 34.2 1383.0)
pmol/L compared to healthy controls levels of 32.1
(range: 11.2 72.3) pmol/L. In the present study,
significantly elevated HE4 concentrations were
found in 28 of 36 cases of ovarian cancer, and cut off
levels of 74 pmol/L gave the sensitivity rate of 78%.
CONCLUSION
Early detection is a critical focus of ovarian cancer

research because of its potential to reduce suffering
and morbidity. HE4 assay values obtained with different assay methods cannot be used interchangeably due to differences in assay methods and reagent
specificity. The results reported by the physician
must include the identity of the HE4 assay used.
The presented results of the analytical evaluation
methods for the determination of HE4 on Elecsys
2010 analyzer showed an acceptable accuracy and
precision.
DISCUSSION
Human Epididymis Protein 4 (HE4) is a 25 KD

single peptide chain with a low expression in epithelia of repertory and reproductive tissues, but highly
expressed in ovarian cancer tissue. HE4 is a novel
serological marker used especially for ovarian cancer
diagnosis because of its high sensitivity (8). The percentage of increase in HE4 values has been used as
an aid in monitoring recurrence or progressive disease in patients with invasive epithelial ovarian cancer. There is no clinically accepted cut-off for use in
monitoring cancer progression in epithelial ovarian
cancer (9). Currently, several biomarker panels are
being evaluated in increase the sensitivity and specificity of ovarian cancer diagnosis. The combination
of CA125 and HE4 has been evaluated to improve
ovarian cancer diagnosis. The data suggests that by
combining these markers the predictive accuracy in
ovarian malignancy is better than by applying any
of the markers alone (10). As a single tumor marker,
HE4 had the highest sensitivity for detecting ovarian cancer, especially in stage I disease.
The analysis of HE4 by competitive chemiluminescent immunoassay on Elecsys 2010 analyser is rapid
and easy to perform. The linearity range of assay is
COMPETING INTERESTS
Authors have no conflict of interest to report.

REFERENCES
1. Ozols RF:Challenges for chemotherapy in ovarian cancer. Ann Oncol
2006; 17 (Suppl.5) 181-187.
2. Fishman DA, Bozorgi K: The scientific basis of early detection of epithelial ovarian cancer: the National Ovarian Cancer Early Detection Program
(NOCEDP), Cancer Treat. Res. 2002; 107:3-28.
3. Ozols RF: Systemic therapy for ovarian cancer: current status and new
treatments. Semin. Oncal. 2006; 33:53-5.
4. Agarwal R, Linch M, Kaye SB: Novel therapeutic agents for ovarian cancer.
Eur J Surg Oncol. 2006; 32:875-886.
5. Kimberly A Lowe, Chiray Shah, Erin Wallace, Garret Andreson, Pamela
Paley, Martin McIntose. Effect of Personal Characteristics on Serum Ca125,
Mesothelin and HE4 Levels in Healthy Post-menopausal Women at HighRisk for Ovarian Cancer, Cancer Epidem Biomarkers 2008;17(9):24802487.
6.
38
Bouchard D, Morriset D, Bourbonnais Y. Proteins with whey-acidic-protein

motifs and cancer. Lancet Oncol 2006;7:167-74.
http://www.jhsci.ba
7. Moore RG, McMeekin DS, Brown AK. A novel multiple marker bioassay
utilizing HE4 and CA125 for the prediction of ovarian cancer in patients
with a pelvic mass. Gynecol Oncol 2009;112:40-6.
9. Allard J, Somers E, Theil R, Use of a novel biomarker HE4 for monitoring of

patients with epithelial ovarian cancer ASCO Annual Meeting Proceedings
2008;26:5535.
8. Galgano MT, Hampton GM, Frierson HF. Comprehensive analysis of HE4

expression in normal and malignant human tissues. Mod Pathol 2006;
19:847-53.
10. Montagnana M, Danese E, Ruzzenente O. The ROMA for estimating the

risk of epithelial ovarian cancer in women presenting with pelvic mass: is it
really useful. Clin Chem Lab Med 2011; 49(3):521-525.
39
Fuad Julardija et al. Journal of Health Sciences 2014;4(1):40-44
http://www.jhsci.ba

RESEARCH ARTICLE
Open Access
Comparison of 3D Maximum intensity projection

(MIP) reconstruction and 2D T2 Half-Fourier
Acquisition Single-Shot Turbo Spin-Echo
(HASTE) sequence in magnetic resonance
cholangiopancreatography
Fuad Julardija*, Adnan ehi, Damir Jaganjac, Esad Voloder, Sreko Maura, Dunja Vrci
Clinic for Radiology, Clinical Center University of Sarajevo, Bolnika 25, 71000 Sarajevo, Bosnia and Herzegovina
ABSTRACT
Introduction: Magnetic resonance cholangiopancreatography (MRCP) is a method that allows noninvasive visualization of pancreatobiliary tree and does not require contrast application. It is a modern method
based on heavily T2-weighted imaging (hydrography), which uses bile and pancreatic secretions as a
natural contrast medium. Certain weaknesses in quality of demonstration of pancreatobiliary tract can
be observed in addition to its good characteristics. Our aim was to compare the 3D Maximum intensity
projection (MIP) reconstruction and 2D T2 Half-Fourier Acquisition Single-Shot Turbo Spin-Echo (HASTE)
sequence in magnetic resonance cholangiopancreatography.
Methods: During the period of one year 51 patients underwent MRCP on 3T Trio system. Patients of
different sex and age structure were included, both outpatient and hospitalized. 3D MIP reconstruction
and 2D T2 haste sequence were used according to standard scanning protocols.
Results: There were 45.1% (n= 23) male and 54.9% (n=28) female patients, age range from 17 to 81
years. 2D T2 haste sequence was more susceptible to respiratory artifacts presence in 64% patients, compared to 3D MIP reconstruction with standard error (0.09), result significance indication (p=0.129) and
confidence interval (0.46 to 0.81). 2D T2 haste sequences is more sensitive and superior for pancreatic
duct demonstration compared to 3D MIP reconstruction with standard error (0.07), result significance
indication (p=0.01) and confidence interval (0.59 to 0.87)
Conclusion: In order to make qualitative demonstration and analysis of hepatobiliary and pancreatic
system on MR, both 2D T2 haste sequence in transversal plane and 3D MIP reconstruction are required.
Keywords: 3D MIP reconstruction, 2D T2 haste sequence, MRCP, pancreatic duct
*Corresponding author: Fuad Julardija

Clinic of Radiology, Clinical Center University of Sarajevo,
Bolnika 25, 71000 Sarajevo, Bosnia and Herzegovina
Phone: +387 33 298 229
E-mail: fudo_78@yahoo.com
INTRODUCTION
Magnetic resonance (MR) imaging allows clear

demonstration of whole body organs, as well as
their pathological changes (1). Magnetic resonance
cholangiopancreatography (MRCP) is the method
2014 Fuad Julardija et al.; licensee University of Sarajevo - Faculty of Health Studies. This
http://www.jhsci.ba
which allows noninvasive visualization of pancreatobiliary tree and does not require contrast agent application (2). It is increasingly being used as a noninvasive radiological method and a high percentage of
the diagnostic results of MRCP are comparable with
those obtained by ERCP for various hepatobiliary
tract pathologies.
Basic principle of MR cholangiopancreatography
is heavily T2-weighted imaging (hydrography) that
uses bile and pancreatic secretions as a natural contrast medium. The current most popular sequences
for MRCP are single-shot fast spin-echo sequences,
which are divided into three types: 2D single slice,
2D multiple slice and 3D methods (3).
Gating is a new addition to MR. It is a process that
allows MR image high resolution, despite motion
presence. Real-time navigator echo gating is a comfortable technique without breath hold that can be
used to compensate various motion types (4). This
technique is applied in MR hepatobiliary and pancreatic systems imaging. In addition to this imaging technique, other imaging techniques can be applied as well, such as breath hold imaging technique.
Miyazaki et al. introduced HASTE (half-Fourier
acquisition single-shot turbo spin-echo) sequences
for acquiring MRCP images. With HASTE acquisitions, Miyazaki et al. were able to generate projection MRCP images using very short scanning time:
2 seconds for the single-slice technique and 18 seconds for the multi-slice technique (5).
Three dimensional (3D) images have increasingly
important role in modern diagnostic radiology.
With program improvement 3D volumetric data
sets can easily be transformed in coronal, sagittal,
oblique or curved cross section planes, which can
help in lesion detection and localization. Maximum
intensity projection (MIP) and multiplanar reconstruction (MPR) are generally used algorithms for
MR cholangiopancreatography (MRCP). MIP allows three dimensional demonstration of biliary
and pancreatic systems. Because of its resemblance
to ERCP images, MIP reconstruction is widely accepted by clinicians. In spite of its usefulness, MIP
may be misleading without a proper reference to
source images or a guidance of MPR. Opacification defects that reflect intra-ductal or intra-cystic
pathologies are notably erased through the process
of MIP reconstruction. Diagnosis based only on

MIP images is therefore not clinically feasible. Use
of any multi-section image is essential, or at least use
of source images (6).
MIP is the projection of highest intensity pixels
onto an arbitrarily oriented plane. MIP images have
an aspect similar to that of conventional angiograms
and are commonly used for angiographic display
such as vascular anatomy evaluation. The drawback
of MIP images is the lack of depth information so
that the objects lying in the same projection plane
of high intensity structures cannot be visualized (7).
The aim of this study was to compare the 3D MIP
reconstruction and 2D T2 (HASTE) sequence techniques in magnetic resonance cholangiopancreatography.
METHODS
Research was conducted at the Clinic for radiology,

Clinical center of University of Sarajevo, during the
period from January to December 2013. The study
included 51 patients of both sex and different age
structure, both outpatient and hospitalized, admitted to a scheduled date of hepatobiliary and pancreatic tract diagnostic imaging. The data for patients
were obtained from their medical records (medical
history, clinician finding sand referral form).
All the patients underwent the examination on the
Trio 3T Siemens system (Siemens, Germany). During the scanning body matrix coil was used. Every
patient underwent standard breath hold T2 haste
and navigator triggering T2 turbo spin echo sequence for MRCP. For T2 haste multibreath hold
sequence the parameters were: Slices 38, Dist. factor
30 %, FoV read 400 mm, FoV phase 60 %, Slice
thickness 5.0 mm, TR 1700 ms, TE 90 ms, TA:1.26
min. Basic resolution 320.
For T2 tse triggering sequence the parameters were:
Slices 86, Dist. factor 30 %, FoV read 380 mm, FoV
phase 100 %, Slice thickness 1.2 mm, TR 1800 ms,
TE 441 ms, TA:4.27 min. Basic resolution 320. After the completed scanning, MIP reconstruction of
hepatic and pancreatic ducts was conducted, where
the hepatobiliary tract ducts and main pancreatic
duct demonstration analysis was done.
41
http://www.jhsci.ba
sented by single admitting diagnosis each (Figure 1).

In the conclusion of the final radiological report
MR findings were generally with no abnormalities
detected 43.2 % (n=22), and the minimum of findings were with choledochal duct dilatation 1.9%
(n=1) and choledochal diverticulosis 1.9% (n=1).
The emphasis on pancreatic duct was in 3.9% (n=2)
patients with pancreatic duct dilatation finding, no
abnormalities were found in other radiological findings. In 5.8% (n=3) cases image analysis was impossible due to respiratory motion artifacts presence
(Figure 2).
In 64% patients 2D T2 haste sequence showed
higher sensibility for the respiratory artifacts presence, that is in this sequence more respiratory artifacts appeared compared to 3D MIP reconstruction
with standard error (0.09), result significance indi-
Descriptive statistics, T-test, Spearman's rank correlation coefficient and ROC curve were used for
data analysis. 3D MIP reconstruction was compared
with 2D T2 haste images in pancreatic duct demonstration resulting in the following.
RESULTS
There were 51 patients, of which 45.1% (n= 23)

male and 54.9% (n=28) female. Patients mean age
was 53 years and ranged from 17 to 81 years. All the
patients stood the examination well and there were
no unwanted effects.
According to admitting diagnosis the patients were
generally diagnosed with cholecystectomy 11.7 %
(n= 6) while in 8 patients other diseases were repre-
FIGURE 1.
Admitting
diagnoses
of the patients
FIGURE 2.
Radiological
findings in the
study group
42
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Based on acquired results it can be concluded that

in pancreatic duct demonstration statistically significant difference exists between 3D MIP reconstruction and 2D T2 haste sequence, on the statistical
significance level pancreatic duct was better demonstrated in 2D T2 haste sequence (p<0.01) compared
to 3D MIP reconstruction, respectively.
DISCUSSION
MRCP is a modern diagnostic radiology method

that offers many possibilities. It is not harmful for
the patients and does not require special preparation,
as in case of ERCP. Its advantages are that MRCP
is noninvasive, cheaper, uses no ionizing radiation,
requires no anesthesia, it is less operator dependent,
better demonstrates ducts proximal to an obstruction or tight stenosisand when combined with conventional T1- and T2-weighted sequences, allows
anatomic imaging of extraductal disease (8).
Apart from its possibilities, there are certain limitations that sometimes can present some sort of pitfalls in radiological finding interpretation. These pitfalls or diagnostic errors may have a variety of causes
and may simulate or mask various diseases of the
pancreatobiliary tract (9).
For the MR imaging, cooperation of patients and
medical radiology engineer performing the imaging procedure is very important because of required
breath hold in some sequences. Respiratory motion
artifact scan present a problem in 3D MIP reconstruction when the patient does not hold the breath
long enough to complete one imaging session. In
our research 2D T2 haste sequence was more sensitive to the respiratory artifacts presence in 64%
patients.
MIP reconstructed images completely obscure small
filling defects due to the partial volume effect (10).
In pancreatic duct demonstration in 3D MIP reconstruction and 2D T2 haste sequence there is statistically significant difference on the statistical significance level (p<0.01)meaning that 2D T2 haste
sequence is more superior than 3D MIP reconstruction in the pancreatic duct examination. Because of
this difference, image analysis acquired by MRCP
needs to include both 3D MIP reconstruction and
2D T2 haste sequence. MRCP can accurately demonstrate the normal pancreatic duct as well as vari-
FIGURE 3. ROC curve for sensibility demonstration of 2D T2

haste sequence for respiratory artifacts presence.
cation (p=0.129) and confidence interval (0.46 to

0.81) (Figure 3).
In 73% patients 2D T2 haste sequence had higher
sensibility for the pancreatic duct demonstration
compared to 3D MIP reconstruction with standard
error (0.07), result significance indication (p=0.01)
and confidence interval (0.59 to 0.87) (Figure 4).
FIGURE 4. ROC curve for sensibility demonstration of 2D T2

haste sequence in pancreatic duct examination.
43
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REFERENCES
ous pancreatic duct abnormalities, including congenital anomalies of the biliary tree and pancreatic
duct (11).
During the imaging when the patient does not
perform an adequate breath hold for the period
required for image acquisition, respiratory artifacts
that degrade 3D MIP reconstruction appearance
can emerge, resulting that choledochal duct and
pancreatic duct may appear stenotic, dilated, or duplicated.
MRCP with a half-Fourier single-shot turbo spinecho sequence depicts not only static fluid in the
pancreatobiliary tree but also slow-flow vascular
structures (e.g. portal vein, hepatic vein) (12) due
to a relatively short echo time. In addition to these
structures 2D T2 haste sequence allows clear morphological demonstration of hepatic and pancreatic
parenchyma, and adjacent structures.
1. Hekimoglu K, Ustundag Y, Dusak A, Erdem Z, Karademir B, Aydemir S, et

al. MRCP vs. ERCP in the evaluation of biliary pathologies: Review of current literature. Journal of Digestive Diseases 2008; 9 (3):162169.
2. Adamek HE, Albert J, Weitz M, Breer H, Schilling D, Riemann JF. A prospective evaluation of magnetic resonance cholangiopancreatography in
patients with suspected biliary obstruction. Gut 1998; 43:680-683.
3. Ohgi K, Furukawa T, Akiyama H, Kimura S, Uehara K, Murata K. Basic
principles and historical consideration of MR cholangiopancreatography.
Nihon Rinsho 1998;56(11):2755-9.
4. Ehman RL, Felmlee JP. Adaptive technique for high-definition MR imaging
of moving structures, Radiology 1989; 173: 255-263.
5. Soto JA. MR Cholangiopancreatography Using HASTE (Half-Fourier Acquisition Single-Shot Turbo Spin-Echo) Sequences. AJR 2007; 189: 5-6.
6. Isogai S, Takehara Y, Isoda H, Kaneko M. Maximum intensity projection
(MIP) and multiplanar reformation (MPR) for post-processing cholangiopancreatographic data set--clinical application and pitfalls. Nihon Rinsho
1998; 56(11):2760-2767.
7. Tongdee R, Narra VR, Brown JJ. Utility of 3D magnetic resonance imaging
in preoperative evaluation of hepatobiliary diseases. HPB (Oxford) 2006;
8(4):311317.
8. Vitellas KM, Keogan MT, Freed KS, Enns RA, Spritzer CE, Baillie JM et al.
Radiologic Manifestations of Sclerosing Cholangitis with Emphasis on MR
Cholangiopancreatography. RadioGraphics 2000; 20(4):959-975.
9. Watanabe Y, Dohke M, Ishimori T et al. Diagnostic pitfalls of MR cholangiopancreatography in the evaluation of the biliary tract and gallbladder.
RadioGraphics 1999; 19:415-429.
CONCLUSION
In demonstration of pancreatic duct 2D T2 haste

sequence had a better result compared to 3D MIP
reconstruction. For MR demonstration and analysis
of hepatobiliary and pancreatic system, it is essential
to use both 2D T2 haste sequence and 3D MIP reconstruction with source images as they supplement
each other.
10. Soto JA, Barish MA, Yucel EK, Ferrucci JT. MR cholangiopancreatography: findings on 3D fast spin-echo imaging. AJR Am J Roentgenol 1995;
165:1397-1401.
11. Ueno E, Takada Y, Yoshida I, Toda J, Sugiura T, Toki F. Pancreatic diseases:
evaluation with MR cholangiopancreatography. Pancreas 1998;16(3):418426.
12. Hennig J, Nauerth A, Friedburg H. RARE imaging: a fast imaging method
for clinical MR. Magn Reson Med 1986; 3:823-833.
COMPETING INTERESTS
The authors declare no conflict of interest
44
http://www.jhsci.ba
Suljo Kuni et al. Journal of Health Sciences 2014;4(1):45-49

RESEARCH ARTICLE
Open Access
Electroneurographic parameters in patients with

metabolic syndrome
Suljo Kuni1*, Emir Tupkovi1,2, Mediha Nii1, Semiha Salihovi1
1
Department of Neurophysiology of Primary Health Centre Tuzla, Albina Herljevia 1, 75000 Tuzla, Bosnia and Herzegovina.
Faculty of medicine, University of Tuzla, Univerzitetska 1, 75000 Tuzla, Bosnia and Herzegovina.
ABSTRACT
Introduction: The aim of this study was to measure electroneurographic (ENG) parameters of the median
and ulnar nerve in patients with metabolic syndrome and to determine whether the large imbalance in
glycemic control came to neuropathic changes to the template.
Methods: The study included 100 patients with metabolic syndrome diagnosed according to the criteria
of the National Cholesterol Education Program - Adult Treatment Panel III (NCEP-ATP III). The patients were
divided into two groups. Group I patients with normal glycemic control and Group II - patients with
diabetes mellitus for up to five years. We measured sensory conductive velocity (SCV), the amplitude of
sensory nerve action potential (SNAP), motor conductive velocity (MCV), terminal motor latency (TML) and
compose muscle action potential after distal stimulation (CMAP-I) and after proximal stimulation (CMAPII) for the ulnar and median nerve.
Results: Sensory and motor parameters in Group II were amended to neuropathic pattern compared to
Group I. There were significant differences in: SNAP amplitude for all tested nerves, SCV values for both
left and right median and ulnar nerve; MCV and TML for left median nerve; MCV, TML and CMAP-I for
right median nerve area; MCV and TML for left ulnar nerve; MCV, CMAP-I and CMAP-II for right ulnar
nerve area.
Conclusion: Patients with metabolic syndrome and diabetes mellitus duration of five years have the
significant changes in sensory and motor peripheral nerves. Neuropathic changes are possible in patients
with metabolic syndrome and normal glycemic control.
Keywords: Electroneurography, Metabolic syndrome, Diabetes mellitus
INTRODUCTION
Metabolic syndrome is a group of risk factors for

cardiovascular disease and type 2 diabetes due to
*Corresponding author: Suljo Kuni
Veljka Lukia Kurjaka do br. 66, 75000 Tuzla,
Bosnia and Herzegovina; Tel: +387 61 67 69 25
e-mail: suljo.kunic@hotmail.com
Submitted December 14, 2014/Accepted March 2, 2014
the existence of abdominal obesity and insulin resistance (1). A criterion for the diagnosis of metabolic
syndrome is quite different among the professional
societies. According to the guidelines of the National Cholesterol Education Program - Adult Treatment Panel III (NCEP-ATP III), obesity is regarded
as the most important criterion for the diagnosis
of metabolic syndrome, defined waist size >102cm
for men and >88cm for women. Other criteria for
2014 Suljo Kuni et al. ; licensee University of Sarajevo - Faculty of Health Studies. This is
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with uremia, hereditary neuropathy or autoimmune

disease with neuropathic symptoms were excluded.
Patients were selected on the basis of data in medical records: information of diabetes mellitus, waist
size, values of blood pressure, serum triglycerides
and high density lipoproteins. Oral glucose tolerance test (OGTT) was used for differentiation of
the level of disorder in glycemic control (9). Based
on the results obtained by OGTT subjects were divided into two groups:
The Group I was comprised of 50 patients (14
males and 36 women) with metabolic syndrome
and normal glycemic control, with a mean age 52.6
years (range 38-65), blood glucose mean level 5.1
mmol/l (range 4 to 5.5 mmol/l), glucose two hours
after OGTT 4.5 mmol/l (range 2.2 to 6.1 mmol/l),
waist circumference 104 cm (range 88-148), systolic blood pressure 134 mmHg (range 110-160),
diastolic blood pressure 88.4 mmHg (range 70100 mmHg), high-density lipoproteins 1.2 mmol/l
(range 0.8 to 1.9 mmol/l), and triglyceride levels 2.8
mmol/l (0.9 to 12.3 mmol/l).
The Group II consisted of 50 patients (19 males and
31 women) who had the metabolic syndrome and
type 2 diabetes mellitus for up to five years, mean
age 61 years (range 38-78 years), fasting blood glucose levels 8.3 mmol/l (range 5.3 to 12.3 mmol/l),
glucose levels two hours after OGTT 11.7 mmol/l
(range 5.4 to 18.2 mmol/l), waist circumference
107 cm (range 85-142 cm), systolic blood pressure
142.3 mmHg (range 100-200 mmHg), diastolic
blood pressure 87.2 mmHg (range 70-120 mmHg),
high-density lipoproteins 1.1 mmol/l (range 0.5 to
2.4 mmol/l), triglyceride levels 3.1 mmol/l (range
0.6 to 12.2 mmol/l).
The study was approved by the Ethics Committee of
the Health Centre Tuzla.
Electroneurographic testing of subjects was conducted at room temperature and "physiological"
skin temperature in a horizontal position with
Electroneurographic Medelec Synergy unit (EMG
and EP Systems, Oxford Instruments 2004, UK).
Superficial stimulating and bipolar registering (so
called Large touchproof ) electrodes were used. Electrostimulation of nerves and registration evoked responses was done according to previously described
diagnosis of metabolic syndrome include dyslipidemia (triglycerides 1.69 mmol/l or high-density

lipoprotein concentrations 1.04 mmol/l for men
and 1.29 mmol/l for women); elevated blood pressure (systolic 130mmHg or diastolic 85 mmHg)
and elevated levels of blood glucose concentration
(6.1 mmol/l or according to different literature
5.6 mmol/l). Three or more criteria or two or more
criteria with obesity defined by waist size are sufficient for the diagnosis of metabolic syndrome (2-4).
Diabetes mellitus is a chronic, incurable systemic
metabolic disorder, which is characterized by permanently elevated levels of glucose in the blood. Mainly conditioned by hereditary factors, and is due to
reduced secretion or reduced biological effects of the
hormone insulin, or the combination of these two
factors. This deficiency interferes with the exchange
of carbohydrates, fats and proteins in the body, and
after a long time affects the structure and function
of blood vessels, nerves, and other vital organs and
organ systems (5, 6).
Peripheral nerve in patients with metabolic syndrome or diabetes mellitus is particularly sensitive to
pressure, stretching or repeated mechanical trauma.
It is assumed that this is a disturbance of nerve recovery in areas normally exposed to damage (7,8).
The aim of this study was to determine the electroneurographic parameters of patients with metabolic
syndrome and normal glycemic control, and in patients who along with the metabolic syndrome have
diabetes mellitus for up to five years, and that based
on the results to check whether a disorder glycemic
control in metabolic syndrome contributes to the
development of neuropathic changes.
METHODS
The study included 100 patients evaluated by teams

of family medicine in Primary Health Centre Tuzla.
Diagnosis of metabolic syndrome was based on laboratory findings and anthropometric measurements,
and taking into consideration criteria of NCEP-ATP
III for diagnosis of metabolic syndrome (2). Patients
with diabetes mellitus type 1, with amputated one
or both upper extremities, patients who had a surgery or injury of deeper tissues and/or who had a tumor localized in the areas of nerves tested, patients
46
http://www.jhsci.ba
standard procedure (10). Electroneurographic

examination included: sensory conductive velocity (SCV) in the hand of the primary deflection
of sensory nerve action potential (SNAP) and the
maximum amplitude of sensory response; motor
conductive velocity (MCV) in the forearm, after the
so-called stimulating artifact to deviations from the
isoelectric line compound muscle action potential
(CMAP), and CMAP areas. Stimulation in determining the motor and sensory responses were done
by stunting amplitude CMAP and SNAP. When
sensory response could not be detected, conductive
speed was considered 0 m/s.
FIGURE 1. Sensory conductive velocity. Median value SCV

for Group I was 49.15 m/s, and for Group II 44.35m/s. The
difference was statistically significant (p<0.001).
Descriptive statistics and Mann - Whitney U - test

were used for data analysis (11). P<0.05 was considered statistically significant. Microsoft Office Excel
2007 and Arcus Quickstat Biomedical software were
used for data processing.
RESULTS
Sensory electroneurographical parameters
In the Group I in 2 (4 %) cases SNAP could not be

induced in the left median nerve, and in 3 (6 %)
patients could not be induced in the right median
nerve. In the Group II SNAP could not be induced
in 4 (8 %) cases for the left median nerve, 2 (4 %)
cases for the left ulnar nerve, 6 (12%) cases for the
right median nerve and 1 (2%) for the right ulnar
nerve. In these cases, the minimum values of the
SNAP amplitude and conduction velocity were documented as 0 m/s and 0 V, respectively. The results
of SCV and SNAP amplitude in both of the groups
of patients, and the differences in their values are
shown in Figure 1 and 2.
FIGURE 2. Sensory nerve action potential amplitudes. Median value SNAP amplitudes Group I was 17.25 V, and for
the Group II 9.30 V. The difference was statistically significant (p<0.001).
Motor electroneurographical parameters
The results show that the neuropathic changes more

prominent in patients with diabetes mellitus, but
that may be encountered in patients with metabolic
syndrome who have normal glycemic control. The
results of MCV and value and CMAP-I areas, and
the differences in their values are shown in Figure 3
and 4. The value of TML and CMAP - II areas are
shown in Figure 5 and 6.
FIGURE 3. Motor conductive velocity. Median MCV value for

Group I was 54.75 m/s, and for the Group II 51.05 m/s. The
differences were statistically significant (p< 0.001).
47
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DISCUSSION
Although they are not immediately recognizable

during the classical neurological examination, neuropathies are common disorders that are following
the metabolic syndrome. Timely insight into these
changes can determine the interim further therapeutic strategy, whose aim should be to prevent further
progression of the syndrome and of its potential destructive effect on the peripheral nerve, which then
may affect the functionality and thus adversely impact on quality of life of patients with this syndrome.
There is little data in the literature about the incidence of neuropathy within the metabolic syndrome
without diabetes mellitus. Smith et al. (2008) have
shown higher incidence of neuropathy in patients
with metabolic syndrome and normal glycemic control compared to the incidence of neuropathy in the
general population. The same study showed a higher
incidence of dyslipidemia in patients with neuropathy than in patients without neuropathy (12).
On the other hand, the huge number of published
studies that confirm the negative impact of diabetes mellitus on the occurrence and development of
neuropathy. Shaw et al. (2003) have shown that the
incidence of diabetic neuropathy increases with age,
duration of diabetes and worse glycemic control
(13), what has been confirmed in our study.
Our research has confirmed once again that the metabolic conditioned changes in the motor nerve conduction, due to a greater imbalance in glycemic control leads to: extending the terminal motor latency,
slow speed motor conductivity (more pronounced
in the ulnar nerves) and reduction in CMAP areas.
All components of the sensory electroneurographical
parameters were significantly altered by neuropathic pattern in the group of patients with metabolic
syndrome and diabetes duration of up to five years.
In a similar survey Said et al. (1983). and Onder et
al. (2012) has been proven that the paresthesia, as a
sign of damage to the sensory nerve function, usually occur as the first symptom of peripheral polyneuropathy and that about 74% of these subjects
had carpal tunnel syndrome (14, 15).
The results and conclusions of this study impose the
need for expansion of protocols of keeping patients
with metabolic syndrome, which includes an obligation neurophysiological testing, for the purpose
FIGURE 4. Compound muscle action potential areas (after

proximal stimulation). Median value CMAP-I areas for Group I
was 19.55 mVms, and Group II 17.15 mVms. The differences
were statistically significant (p = 0.0032).
FIGURE 5. Terminal motor latency. Median value TML for

Group I was 3.10 m/s, and Group II 3.38 m/s. The difference
was statistically significant (p<0.001).
FIGURE 6. Compound muscle action potential areas (after

distal stimulation). Median value CMAP-II areas Group I was
17.80 mVms, and Group II 15.25 mVms. Differences were
statistically significant (p = 0.0018).
48
http://www.jhsci.ba
2001; 285:2486-97.
of early identification and appropriate therapeutic

treatment of these patients.
3. Deen D. Metabolic Syndrome: Time for Action. Am Fam Physician 2004;

69:2875-82.
4. Diagnosis and management of the metabolic syndrome: an American
Heart Association/National Heart, Lung, and Blood Institute Scientific
Statement. Circulation 2005; 112:2735-52.
CONCLUSIONS
Patients with metabolic syndrome and diabetes

mellitus duration of five years have the significant
changes in sensory and motor peripheral nerves.
Neuropathic changes are possible in patients with
metabolic syndrome and normal glycemic control.
5. Birder LA, Perl ER. Cutaneous sensory receptors. J Clin Neurophysiol.

1994 Nov;11(6):534-52.
6. Lee DH, Claussen GC, Oh S. Clinical nerve conduction and needle electromyography studies. J Am Acad Orthop Surg. 2004;12(4):276-87.
7. Barada A, Reljanovi M, Bili R, Kovljani J, Metelko . One year follow up
in diabetic patients after surgical treatment of carpal tunnel syndrome. J
Neurol 2000; 247(3):753.
COMPETING INTERESTS
8. Moghtaderi A, Ghafarpoor M. The dilemma of ulnar nerve entrapment at

wrist in carpal tunnel syndrome. Clin Neurol Neurosurg 2009; 111:151-5.
The authors declare no competing interests.
9. International Association of Diabetes and Pregnancy Study Groups Consensus Panel. Diabetes Care. 2010 Mar;33(3):676-82.
10. American Diabetes Association Consensus statement: standardized measures in diabetic neuropathy. Diabetes Care 1995 ; 18: Suppl 1: 59-82.
ACKNOWLEDGMENTS
11. Petz B. Osnovne statistike metode za nematematiare, Naklada Slap,

Jastrebarsko; 1997: 327-328.
We thank dr. Avdurahman Kuni for his cooperation.
12. Smith AG, Singleton JR. Impaired glucose tolerance and neuropathy. Neurologist 2008; 14(1):23-9.
13. Shaw JE, Zimmet PZ, Gries FA, Ziegler D. Epidemiology of diabetic neuropathy. In: Gries FA, Cameron NE, Low PhA, Ziegler D, editors. Textbook
of diabetic neuropathy. Stuttgart: Thieme, 2003; 64-79.
REFERENCES
1. Blaha MJ, Bansal S, Rouf R, Golden SH, Blumenthal RS, Defilippis AP. A
practical ABCDE approaach to the metabolic syndrome. Mayo Clin Proc
2008;83:932-41
14. Said G, Slama G, Selva J. Progressive centripetal degeneration of axons

in small fibre diabetic polyneuropathy. Brain 1983; 106:791-807.
2. Executive Summary of The Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA
15. Onder B,Yaln E,Seluk B,Kurtaran A,Akyz M. Carpal tunnel syndrome

and metabolic syndrome co-occurrence. Rheumatol Int. 2013; 33(3):583-6
49
Haris Vrani et al. Journal of Health Sciences 2014;4(1):50-54
http://www.jhsci.ba

RESEARCH ARTICLE
Open Access
The incidence of vascular complications after

coronary angiography: evaluation of results
and risk factors
Haris Vrani*, Ilirijana Haxhibeqiri-Karabdi, Amel Hadimehmedagi
Institute for heart diseases, University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina
ABSTRACT
Introduction: The aim of this study was to present the incidence of the vascular complications that had
to be surgically treated during the two-year period of transfemoral cardiac catheterization procedure and
to identify the risk factors associated with the complications.
Methods: A retrospective two-year study of post-catheterization complications with the six-month postoperative follow-up and analysis of risk factors was done. Patients with cardiovascular diseases who underwent therapeutic or diagnostic coronary angiography in the period of 2012-2013 were included in the
study. A total of 1320 patients were subjected to catheterization for coronary angiography, of which 24
had vascular complications that had to be surgically treated. Indications for operative treatment included
rapid growth of pseudoaneurysm, hemorrhage, large hematoma, hemodynamic instability, failure of the
targeted compression therapy.
Results: Twenty-four patients experienced some kind of post-operative complication. Infection and dehiscence of surgical wound were the two most common complications. There were no fatalities. The average
length of a hospitalization was 4 days. The important risk factors are gender (women more than man),
obesity, concomitant use of anticoagulation therapy and antiplatelet therapy after catheterization.
Conclusion: Insufficient length of the compression of the punctured place and increased risks of a pseudoaneurysm formation, such as female gender, obesity, and use of a combined anticoagulant therapy are
the main causes of these complications. Late vascular complications are not uncommon.
Keywords: pseudoaneurysm; cardiac catheterization; postoperative complications, risk factors
INTRODUCTION
Pseudoaneurysm represents a constant rupture of

all layers of the arterial wall wherein arterial blood
*Corresponding author: Haris Vranic
Institute for heart diseases, UKC Sarajevo
Bolnicka 25, 71000 Sarajevo, Bosnia and Herzegovina
Phone: +387 61 55 04 00
E-mail: harisvranic@bih.net.ba
is constantly pouring out into the environment by

creating a pseudo bag which is a result of a defense
mechanism of the surrounding tissue. Pseudoaneurysm usually occur after catheterization, at the site
of anastomosis native arteries and synthetic graft,
after trauma, and very rarely, after an infection (e.g.
fungal PSA) (1). The resulting punctured lesions of
femoral artery after intervention do not close, but
rather, extravasation of blood into the surrounding
Submitted March 5, 2014 / Accepted April 20, 2014
2014 Haris Vrani et al.; licensee University of Sarajevo - Faculty of Health Studies. This is
http://www.jhsci.ba
was done every hour until complete removal of local

compression (bags with 750 grams of sand), and the
next day the examination was carried out every three
hours. Diagnosis was usually conducted easily, and
it was a combination of a physical examination and
ultrasound findings.
The ultrasound diagnostic criteria were: the presence
of cavity on B-mode of a ultrasound, pulsating expansion and contraction of area and two-way flow of
blood to the neck of the pseudoaneurysm in systole
and diastole. CT angiography was indicated in patients with acute ischemia of the lower limbs.
Indications for surgical treatment of vascular complications were: rapidly increasing of the pseudoaneurysm, very large hematoma, a drop in blood
counts, acute ischemia of a leg caused by local blood
flow interruption due to dissection or due to compressive action of the pseudoaneurysm on the femoral artery, neuropathy caused by local pressure on
the femoral nerve, threatened viability of the soft tissue (skin necrosis), the failure of digital compression.
All patients, who were included in the study, had
the control of the local status after 3 months, and all
surgically treated patients had control after 3 and 6
months of intervention.
perivascular space occurs, resulting in hematoma

and pseudoaneurysm wall.
Post-catheterizational pseudoaneurism is one of the
most common vascular complications after this diagnostic examination. Its incidence on diagnostic
catheterization ranges from 0.05% to 2%, but after
coronary or peripheral interventions, this percentage is growing from 2% up to 6% (2). The pseudoaneurysm formed on such way often undergoes
thrombosis, spontaneously within a few weeks,
however, those larger than 1.8 cm in diameter or
pseudoaneurysm occurring in patients treated with
anticoagulation therapy usually has to be surgically
treated (3). The smaller caliber of arteries which is
specific for the female gender, as well as difficult
catheterization and insufficient post-procedural
compression of the arteries in obese patients are the
main cause of frequent complication (4).
The aim of this study was to present the incidence of
the vascular complications that had to be surgically
treated during the two-year period of transfemoral
cardiac catheterization procedure and to identify the
risk factors associated with the complications.
METHODS
A retrospective study included patients with vascular

complications of pseudoaneurysm after transfemoral coronary angiography performed at the Clinical
Center of Sarajevo, Institute for heart diseases, during the period from 2012 to 2013. All the patients
underwent retrograde percutaneous transfemoral
catheterization of heart for diagnostic or therapeutic purposes. The study included only patients
with post-catheterizational complications of femoral artery. For each patient age, sex, BMI, type of
catheterization, duration of catheterization, types of
complications (thrombosis, infection,
hemorrhage, pseudoaneurysm, etc.), length of compression, time period when complications occurred,
the length of stay in hospital and clinical outcome
was documented.
After catheterization digital compression was performed during 15 minutes. Size of the sheat was
5F for diagnostic catheterization, and 6F or 7F for
therapeutic catheterization. All patients were hospitalized for at least one day after the procedure. In
all patients a clinical examination of the local status
Statistical analysis was performed by using SPSS

14.0 (Chicago, IL, USA). The level of significance
was set to P<0.05.
RESULTS
Of the 24 patients treated, 19 were women and 5

were men. The median age was 72 years (range 4282 years). Femoral pseudoaneurysm after cardiac
catheterization, which was diagnosed on ultrasound,
was observed in 59.8% of cases. CT angiography
was performed in 11.4%. Physical findings were significant in 13.2% of the patients.
It was observed that the complications were more
associated with the patients with hypertension and
high BMI, but the differences were not statistically
significant (Table 1).
Pseudoaneurysms of femoral artery were present in
60% of cases. Hematoma was found in 26%, arterial dissection was found in 8% of cases, bleeding
51
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TABLE 1. Risk factors associated with complications

Variable
Control group
(n=1296)
Age
Hypertension
BMI (kg/m)
71.3 9.9
712 (54.0%)
24.85 3.12
With
complications
(n=24)
70.11 2.8
19 (77.3%)
28.69 3.33
The patients with vascular complications and subsequent surgery were in 64.2% subjected to diagnostic
and 35.8% to therapeutic procedure. Distribution
of complications during the year was interesting as
most of them occurred in the summer months, and
less in the spring and autumn, while in winter almost no cases of complication were found.
P value
0.378
0.102
0.692
BMI, body mass index
DISCUSSION
Large studies that have dealt with the incidence of

post-catheterizational vascular complications have
come up with results that complications occur in
the range of 0.3% to 1.0%. In our study, such incidence was 1%. Kresowik et al, in the study of 144
patients with control ultrasound after
the therapeutic catheterization, came to
the incidence of vascular complications
TABLE 2. Cumulative results of detected vascular complications
of 9% of patients (5).
Similar results have been reported by
Age and
Time of
Messina
et al. in a four-year study in
Patient
Symptom
Diagnostic Diagnosis
gender
onset*
which there were 106 surgically treat1
58 M
pulsating mass 1
US
PSA
ed patient for vascular post-catheter2
61 F
pulsating mass 2
US
PSA
izational complication, observed that
3
67 M
Hematoma
1
None
Hematoma
prolonged local hemorrhage leads to
4
71 M
pulsating mass 2
US
PSA
the occurrence of a large number of
5
59 F
bleeding
1
CTA
bleeding
pseudoaneurysm (6). In our study, the
6
60 F
pulsating mass 3
US
PSA
size of sheath lead was between 6 and
7
65 F
pulsating mass 2
US
PSA
7 F, but we could not prove any con8
70 F
Leg ischemia
1
CTA
Artery dysection
nection between the size of lead and
9
78 F
pulsating mass 3
US
PSA
occurrence of complications. Although
post-catheterizational complications
10
54 M
Hematoma
4
None
Hematoma
are previously treated only via surgery,
11
62 F
pulsating mass 2
US
PSA
now in modern practice more conser12
84 F
pulsating mass 12
US
PSA
vative treatment are administered by an
13
59 M
Hematoma
4
None
Hematoma
application of a thrombin or a target
14
60 M
pulsating mass 2
US
PSA
compression under ultrasound (7, 8).
15
68 F
pulsating mass 2
US
PSA
16
67 F
Hematoma
5
None
Hematoma
Pseudoaneurism is the most common
17
52 M
Leg ishemia
1
CTA
Artery desection
complication in our study. Clinical di18
58 F
pulsating mass 3
US
PSA
agnosis of the pseudoaneurysms of the
19
84 F
Hematoma
3
None
Hematoma
femoral artery in most cases was obvi20
59 M
pulsating mass 2
US
PSA
ous and easy to diagnose. However, for
21
72 M
Leg ishemia
1
CTA
Foreign body
all patients we have also performed the
Duplex ultrasound, which is the meth22
68 F
Hematoma
4
None
Hematoma
od of choice for the assessment of its
23
48 F
pulsating mass 3
US
PSA
size, the thrombotic material inside the
24
47 F
pulsating mass 62
US
PSA
bag, and for clearly specifying the size
*Days after catheterization procedure; US, ultrasound; CTA, Computed tomogand speed of communication with the
and foreign body in 3% of cases. Postoperative

complications were found in 8 cases: dehiscence of
a wound in 3 cases, or 39%, deep vein thrombosis
in one case or 11%, and infection in 50% of cases
(Table 2).
raphy angiography; PSA, Pseudoaneurism; M, male; F, female
52
http://www.jhsci.ba
CONCLUSION
artery (9). In our study, the use of targeted compression at pseudoaneurysm under the control of
the ultrasound was effective in all cases with smaller
neck pseudoaneurysm (up to 3 mm), but those with
larger necks and rapid flow through it, there was no
effect. Similar results were received by Fellmeth and
Eisenberg in their studies conducted in over 1500
subjects in which they are all treated with targeted
ultrasound compression of the femoral artery bleeding. (10). From our experience, and the obtained
results, we have achieved good results with extended
digital compression with minimal lowering of blood
pressure and strict bed rest which is consistent with
research completed by Dangasa and colleagues (11).
The incidence of deep vein thrombosis in our study
was 0.5% which is in line with research conducted
by Davis and others (12). However, the incidence of
asymptomatic venous thrombosis is unknown and
must be determined in a future, of a targeted prospective study.
Acute ischemia is in all cases treated surgically. There
are estimated two different types of acute ischemia:
in the two cases there was an acute thrombosis of the
femoral artery at the site of catheterization due to
dissection of atheromatous plaque. In one case, the
reconstruction of arteries was performed with reinsection of the profunda femoris artery, and another
patient underwent femoropopliteal reconstruction.
In both cases the Dacron prosthesis was used. The
third case was a dissection of iliac artery with Terumo guide where iliac-femoral reconstruction was
used. Similar complications mentions Schneinert in
his retrospective study (13). Fortunately, we had no
loss of limbs in surgical patients, although amputations after these complications are not that rare (14).
Mechanisms of complications after catheterization
depend on the proper technique of catheterization,
especially in atherosclerotic changed arteries, on the
manner and duration of compression, controlling
hypertension, and concomitant use of various antithrombotic medications.
However, our results show that complications can
occur after a long period, even after several months
(62 days) (15). We have also come to the results that
indicate that complications are more common in
women, especially those who are obese, with a high
BMI which was confirmed by Applegate et al. (16).
Vascular complications after cardiac catheterization

are rare, but if undetected they can become compromising for limbs and life-threatening. Early clinical
detection and timely consultation with a vascular
surgeon are of paramount importance.
COMPETING INTERESTS
The authors declare no conflict of interest.

REFERENCES
1. Skillman JJ, Kim D, Baim DS. Vascular complications of percutaneous
femoral cardiac interventions. Arch Surg 1988;123:12071212.
2. Coley BD, Roberts AC, Fellmeth BD, Valji K, Bookstein JJ, Hye RJ. Postangiographic femoral artery pseudoaneurysms: further experience with US
guided compression repair. Radiology 1995;194:307311
3. San Norberto Garca EM, Gonzlez-Fajardo JA, Gutirrez V, CarreraS,
Vaquero C. Femoral pseudoaneurysms post-cardiac catheterization surgically treated: evolution and prognosis. Interact Cardiovasc ThoracSurg
2009;8:3537.
4. Oweida SW, Roubin GS, Smith RB 3rd, Salam AA. Poscatheterization vascular complications associated with percutaneous transluminal coronary
angioplasty. J Vasc Surg 1990;12:310315.
5. Kresowik TF, Khoury, Miller BV, Winniford, Shamma AR, et al. A prospective study of the incidence and natural history of femoral vascular complications after percutaneous transluminal coronary angioplasty. J Vasc Surg
1991;13:328333.
6. Messina LM, Brothers TE, Wakefield TW, et al. Clinical characteristics and
surgical management of vascular complications in patients undergoing cardiac catheterization: interventional versus diagnostic procedures. J Vasc
Surg 1991;13:593600.
7. Morgan R, Belli AM. Current treatment methods for postcatheterization
pseudoaneurysms. J Vasc Interv Radiol 2003;14:697710.
8. Sprouse L, Botta D, Hamilton I: The management of peripheral vascular
complications associated with the use of percutaneous suture-mediated
closure devices. J Vasc Surg 2001; 33:688-693.
9. Coley BD, Roberts AC, Fellmeth BD, Valji K, Bookstein JJ, Hye RJ. Postangiographic femoral artery pseudoaneurysms: further experience with
US-guided compression repair. Radiology. 1995;194:307311
10. Cheri Davis, Sharon Vanriper, Jennifer Longstreet, et al. Vascular complications of coronary interventions.Heart & Lung: The Journal of Acute and
Critical Care. Volume 26, Issue 2, MarchApril 1997, Pages 118127
11. Dangas G, Mehran R, Duvvuri S, Ambrose JA, Sharma SK. Use of compression as a treatment option for femoral artery pseudoaneurysms after
percutaneous cardiac procedures. Cathet Cardiovasc Diagn. 1996;39:138
142.
12. Applegate RJ, Sacrinty MT, Kutcher MA, et al. Propensity score analysis of
vascular complications after diagnostic cardiac catheterization and percutaneous coronary intervention using thrombin hemostatic patch-facilitated
manual compression. J Invasive Cardiol 2007;19:164 70.
13. Dierk Scheinert, Josef Ludwig, Herrmann J. Steinkamp, Malte Schrder,
Joern O. Balzer, and Giancarlo Biamino, Treatment of Catheter-Induced
Iliac Artery Injuries With Self-Expanding Endografts. Journal of Endovascular Therapy: June 2000, Vol. 7, No. 3, pp. 213-220.
14. Tavris DR, Gallauresi BA, Lin B, et al. Risk of local adverse events following
cardiac catheterization by hemostasis device use and gender.J Invasive
Cardiol 2004;16:45964.
53
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15. Puri, Vinod K. Carlson, Richard W. Bander, Joseph J. Weil, Ax Harry. Complications of vascular catheterization in the critically ill: A prospective study.
Critical Care Medicine: September 1980
16. Applegate RJ, Sacrinty MT, Kutcher MA, et al. Vascular complications
in women after catheterization and percutaneous coronary intervention.1998-2005. J Invasive Cardiol 2007;19:37537
54
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Edina Tanovic et al. Journal of Health Sciences 2014;4(1):55-58

RESEARCH ARTICLE
Open Access
The effect of the infection Clostridium difficile on

the rehabilitation
Edina Tanovic1*, Haris Tanovic2, Aldijana Kadic1, Devad Vrabac1,
Senad Selimovi3, Dragan Kosti4
1
Clinic for Physical Medicine and Rehabilitation, Clinical Center University of Sarajevo, Bosnia and Herzegovina. 2Clinic for
Abdominal Surgery, Clinical Center University of Sarajevo, Bosnia and Herzegovina. 3JZU BRC Aquaterm, Olovo, Bosnia and
Herzegovina. 4Clinical Center University of Banja Luka, Bosnia and Herzegovina.
ABSTRACT
Introduction: Clostridium difficile is the cause of the post antibiotic colitis. This anaerobe, sporogenous,
gram-positive bacteria is most often recognized as the cause of the nosocomial diarrhea. The aim of this
work is to show the impact of the infection Clostridium difficile on the result of rehabilitation of the patients that have been treated in the rehabilitation facility.
Methods: 448 patients treated at the Clinic for physical medicine and rehabilitation of the Clinical Center
University of Sarajevo were included in the study. Gender, age, Barthel index, length of hospitalization,
and values of the albumin in the serum were documented. Kolmogorov-Smirnov test, Mann-Whitney U
test and One Sample Wilcoxon Signed Rank test were used for data analysis.
Results: There were 57% female and 43% of male patients. The average age was 67.5 years for women
and 52 years for men. Barthel index at admission was 4.0 and at discharge raised to 8.0 (p=0.047). The
length of the hospitalization for patients without infection was shorter (28.8 days) compared to patients
with infection (43 days) (p=0.015). Values of the albumin in the blood at patients with confirmed Clostridium difficile infection were significantly lower than referent values (p = 0.016).
Conclusion: Patients with Clostridium difficile infection had longer period of the rehabilitation and the
results were less favorable.
Keywords: Clostridium difficile, Barthel index, rehabilitation
INTRODUCTION
Clostridium difficile is the cause of the post antibiotic colitis. This anaerobe, sporogenous, gram-positive bacteria is most often recognized as the cause of
the nosocomial diarrhea (1-4).
*Corresponding author: Edina Tanovic,
Clinic for Physical medicine and rehabilitation, Clinical
Center Universitiy of Sarajevo, Bosnia & Herzegovina,
E-mail: tanovicedina@hotmail.com
Diarrhea caused by the Clostridium difficile can either occur individually or in smaller epidemics and
can be transmitted from person to another person
(5,6). It occurs up to 8% of the hospitalized patients
and can be responsible for 20-30% cases of diarrhea. Risk factors are advanced age, serious illnesses,
lengthy period of hospitalization, residence at nursing homes or chronic patients (7-9). Lately, it has
been proven also that risk factors include application of blockage of proton pump and application
of non-steroid anti-rheumatic (10). Dominant pre-
2014 Edina Tanovic et al.; licensee University of Sarajevo - Faculty of Health Studies. This
http://www.jhsci.ba
disposed factor of the host is the antibiotic induced

gastro intestinal disorder. The course of the disease
varies from asymptomatic to necrosis colitis (11-13).
Rarely there are limited impacts of the tissue as well
as sepsis of the acute abdomen. With previous application of the cephalosporin there can be occurrences
of not fully formed stool (not liquid) and leukocytes
in the stool (14,15).
Asymptomatic patients with Clostridium difficile
in their stool are more common than symptomatic
patients, in ratio 3:1. After diarrhea caused by Clostridium difficile, reactive arthritis is described (1618). Risk factors are: advanced age, serious illnesses, lengthy period of hospitalization, residence at
nursing homes or chronic patients. Typical clinical
symptoms are diarrhea, increased body temperature
(fever), pain in the stomach area and leucocytosis
(19,20). The cases of ileus are increasingly described,
as well as cases of perforation of colon, toxic megacolon with development of the shock, to very com-
plicated colitis that can lead to death (21,22).

The aim of this work is to show the impact of the
infection Clostridium difficile on the result of rehabilitation of the patients that have been treated in
the rehabilitation facility.
METHODS
Total of 448 patients were hospitalized at the Clinic

for physical medicine and rehabilitation of the Clinical Center University of Sarajevo, in the period of
May 2011 to May 2012. As the source of the information, we have used the existing medical documentation. We have done ELISA RIDASCREEN
Clostridium difficile to determine the existence of
the toxin in the stool. We analyzed the following:
gender, age, results of the rehabilitation with Barthel
index, length of the hospitalization, as well as values
of the albumin in the blood.
Kolmogorov-Smirnov test, Mann-Whitney U test

and One Sample Wilcoxon Signed Rank test were
used for data analysis. P<0.05 was considered statistically significant.
RESULTS
Infection with C. difficile occured 7 (2%) out of total 448 patients. Of those, 4 (53%) were female and
FIGURE 1. Age distribution in patients with C.difficile infection
FIGURE 3. Length of hospitalization for patients with Clostridium difficile infection compared to patients without infection with mean length of hospitalization of 28.8 days (Z=2.282;
P=0.015).
FIGURE 2. Barthel index at admission and discharge (Z=2.120; P=0.047).

56
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cur at younger patients as well. The majority of cases

of infection developed with 30 days of admission
(27).
The results of the rehabilitation were estimated using Barthel index. All of the suspected patients were
isolated and the process of the rehabilitation at the
proven cases was conducted in special conditions.
I our work we noticed significant statistical difference in the values of Barthel index at the admission
(M=4.0) and discharge (M=8.0), p=0.047. However, results of the rehabilitation with patients with
C.difficile are less effective compared to patients
without infection. Similar results were obtained by
Orisini et al. The hospitalization for patients without
infection with C.difficile is shorter (M=28.8) compared to patients with infection (M=43.0). Similar
results were verified by Orsini et al. which note the
length of the hospitalization at 55.3 days.
The values of the albumin in the blood for patients
with infection C.difficile are significantly lower
than the referent values; there is a high significance
p=0.016. Several research studies show the same or
similar results with differently pronounced values
of the significance. We have not verified horizontal
transmission of the infection. Measurements for the
control of the infection are important for reducing
the transmission of the C.difficile from health workers to patients, as well as between the patients.
Reduced used of the clindamycin in the hospitals,
has reduced the incident of the illness. Relapse can
occur in 15-20% of the patients.
Higher occurrence of the illness caused by Clostridium difficile can be interpreted with the selection
of the new type NAP1/027 which excretes much
higher levels of the toxins A and B compared to until now prevailing types of the Clostridium difficile.
It is assumed that the gene mutation occurred which
regulates the secretion of the toxins. It is still unclear how much effect on the selection of this type
of Clostridium difficile had the prescribing as well as
use of anti-microbic medicine.
Importance of the CD following is also indicated by
Olson et al. who examined the mortality connected
with the infection of the C.difficile. Their results on
908 cases of CDAD show that 6 patients (0.06%)
died from the active pseudomembranosis colitis
(PMC) as the primary disease.
FIGURE 4. Albumin in the blood at patients with C.difficile

(Mean=29.0 g/L; IQR=26.0 to 31.0) compared to referent values (53.0-64.0 g/L) (Z=-2.281; P=0.016).
3 (47%) were men. The mean age for females was

67.5 years and for males 52.0 years (Figure 1). The
Barthel index values were significantly different at
admission and discharge (Figure 2). Also, the length
of hospitalization was sigificantly different for patiens with and patients without C.difficile infection
(Figure 3).
DISCUSSION
In our study we analyzed 448 patients. With 11

patients, there was a suspicion of the infection
C.difficile and at 7 patients, the infection was confirmed. All the patients were accepted for the rehabilitation as transfers from the Clinic for Neurology.
The frequency of the infection with Clostridium difficile in our study was 2%. The research of the Orsini et al. published in 2010 on included 747 patients
with 22 having infection, with frequency of 2.9%.
Female patients were slightly more represented in
the study (57%) which does not correspond with
other research which demonstrates that male genders are more represented.
When we analyze age of the patients, we see different results. In literature we found different values
depending on the institution where the research was
conducted. The average patients were represented at
the advanced age but the illness can sporadically oc57
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8. Bignardi GE. Risk factors for Clostridium difflcilc infection. J Hosp Infect
1998:40: 1-15.
Results of the study done by Morris et al. show

that the mortality of the patients with colonotomy,
toxical megacolon and perforatated colon was high
(up to 50%). In the United Kingdom hospitals it
is noted the increase of death cases that had infection C.difficile during their primary disease. The
information also indicates two to three times higher
death rate during year 2004 compared to year 1999
(20). Prevention remains a priority, high standards
of hygiene and careful attention to the judicious
prescription of antibiotics and crucial regard (28).
Patients admitted to acute rehabilitation may have
an elevated rate of intestinal colonization with C.
difficile without clinical symptoms. Inadvertent
transfer of the organism within the rehabilitation
setting may occur because asymptomatic colonization is not recognized (29). Effective management
requires a coordinated effort among all members of
the healthcare team to facilitate early identification
of patients at risk for CD, early recognition of disease onset and confirmatory testing, prompt initiation of the most appropriate management approach
and ongoing monitoring throughout the continuum care (19,23,30).
.
9. Thibault A Miller MA. Gaese C. Risk factors for thc development of Clostridium diffcile-associated diarrhea during a hospital outbreak. Infect Control
Hosp Epidemiol 1991: 12:345-8.
10. Schwaber MI. Simhon A. Block C. Roval V. Ferderber N, Shapiro M. Factors associated with nosocomial diarrhoea and C. difficile associated
disease on the adult wards of an urban tertiary care hospital. Eur .I Clin
Microbiol Infect Dis 2000;19:9-15.
11. Delmee M, Vandercam B. Avesani V. Epidemiology and prevention of Clostridium difficile infection in leukemia unit. Eur J Clin Microbiol 1987:6:623-7.
12. Watanakunakorn PW. Hazy R. Risk factors associated with Clostridium difficile in hospitalized adult patients: a casecontrol study. Infect Control Hosp
Epidemiol 1996: 17:232-5.
13. Samore MI-I, Venkataraman L Det.iirolami PC, Arbc:it RD, Karchmcr AW.
Clinical and molecular epidemiology or sporadic and clustered cases of
nosocomial Clostridium diffciie diarrhea. Am .J Med 1996: I00:32-40.
14. Spencer RC. Clinical impact and associated costs of Clostridium dificileassociated disease. 1 Antirnicrob Chernether 1998:41:5-12.
15. DuPont HL. Ribner BS, Bennett lV. Hospital infections. 3,dcd. In: Infectious
gastroenteritis. Boston: Little Brown: 1992:641-58.
16. Olson MM. Shanholtzer Cl. Lee JT. Gerding DN. Ten years of prospective
Clostridium difficile disease surveillance and treatment at the Minneapolis VA
Medical Center. 1982 -91. Infect Control Hosp Epidemiol 1994; 15:371 -81.
17. Bowen KE. Mcfarland LV. Greenberg N. Ramsey MM, Record KE. Svenson
J. Isolation of Clostridium difficile at a University hospital: a two-year study.
Clin Infect Dis 1995:20:261-2.
18. Beaugerie L, Flahaut A. Barbut F. Diarrhea des antibiotiques et Clostridium
difficile en population generalc. In:.Iournees Francophones de pathologies
digestive. Paris: 2001:1-14.
19. McFarland LV, Mulligan ME. Kwok RYY. Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 1989:320:204-10.
CONCLUSION
20. Morris B. Yollinger RM. Stellato TA. Role of surgery in antibiotic-induced

pseudomembranous enterocolitis. Am .ISurg 1990:160:535-9.
Patients that had infection with Clostridium difficile had longer period of the rehabilitation and the
results were less favorable.
21. Deaths ivolving Clostridium difficile: England and Wales. 1999-2004.

Health Statistics Q2006:30:56-60.
22. Berg RJ. Kuijper EJ. Claas EC.I. Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR. Clin
Microbiol Infect Dis 2006:12: 178-96.
23. Wongwanich S. Pongpcnch P. Dhirapurta C. Characteristics of Clostridium
difficile strains isolated from asymptomatic individuals and from diarrheal
patients. Clin Microbiol Infec Dis 2001:7:438-4.
COMPETING INTERESTS
Authors declare no conflict of interest.
24. Gayane M. Szczesny A. Silva L. Clostridium difficilc in emergency room.

Clin Microbiol 2005;11:258-61.
REFERENCES
25. Gayane M, Stuart C. Szczesny A. Silva J. Analysis of Clostridium difficileassociatcd diarrhea among patients hospitalized in tertiary academic hospital. Diagn Microbiol Infect Dis 2005;52:153-5.
1. Wilson KH. The microecology of Clostridium difficile.Clin Infect Dis

1993;16(SuppI4):214-8.
26. Orsini S, Lecchi C, Mule C, Maestri A, Astolfi N, Taveggia G. Impact of Clostridium difficile infection in a Rehabilitation unit. In 17th ESPRM Europian
Congress pf Physical and rehabilitation medicine. Edicioni Minerva Medica
Turin 2010; 104-8.
2. McFarland LV, Stamm WE. Review of Clostridium difficile associated diseases. Am J Infect Control 1986: 14:99-109.
3. Borriello Sl'. Pathogenesis of Clostridium difficile infection. I Antimicrob
Chemother 1998:41 (Suppl C): 13-9.
27. Aslam S. Treatment of Clostridium difficilc-associatcd disease: old therapies and new strategies. Lancet Infect Dis 2005:5:549-57.
4. Bartlett IG. Chang TW, Gurwith M. Gorbach SL, Onderdonk AB. Antibioticassociated pseudomembranous colitis due to toxin-producing clostridia. N
Engl J Med 1978;298:531-4.
28. Gastillon BO, Harbath S, Pacudki V. Clostridium difficile infections, specific

issues in the elderly.Rev Med Suisse 2013;9:2044-9.
5. Samore MH. Epidemiology of nosocomial Clostridium difficile infection.

Compr Ther 1993; 19: 15 I -6.
29. Marciniak C, Chen D, Stein AC, Semik PE. Prevalence of Clostridium difficile colonization at admission to rehabilitation. Arch Phys Med rehabil
2006;87(8): 1089-90.
6. Kuijper EJ. Coignard B. Emergence of Clostridium difJicile-associated disease in North America and Europe. Clin Microbiol Infect Dis 2006; 12:2-18.
30. Mylotte JM, Russell S, Sacket B, Vallone M, Antalek M. Surveillance for

Clostridium difficile infections in nursing homes. J Am Geriatr Soc 2013;
61(1): 122-5.
7. Barbut F. Epidemiology of Clostridium difficile-associated infections. Clin

Microbiol Infect 200 1:7:405-11.
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Senka Mesihovi Dinarevi, Emina Vukas Journal of Health Sciences 2014;4(1):59-62

CASE REPORT
Open Access
Cardiac aspects of DiGeorge syndrome: a report of

two cases with molecular analysis
Senka Mesihovi Dinarevi, Emina Vukas*
Pediatric Clinic, Clinical Centre University of Sarajevo, Patriotske lige 81, 71000 Sarajevo, Bosnia and Herzegovina
ABSTRACT
DiGeorge syndrome (DGS) which is also known as velocardiofacial syndrome is caused by a submicroscopic chromosome deletion of band 22q11. It is associated with a disturbed development of the pharyngeal arches. In this report we describe two unrelated male children with clinical features consistent
with 22q11.2 microdeletion syndrome characterized by cardiac defect, recurrent respiratory infections
and developmental deficiency. Definitive diagnosis is made by Fluorescence In Situ Hybridization analysis
(FISH). Children underwent surgical correction of congenital heart defects. During surgery thymic aplasia
was confirmed in both children, postoperative course proceeded without major complications. Our report
suggests that the criteria in searching for microdeletion 22q11.2 should be expanded and applied in patients with conotruncal and non-conotruncal congenital heart defects and at least one typical feature of
this syndrome.
Keywords: DiGeorge syndrome, congenital heart disease, microdeletion
INTRODUCTION
Congenital heart disease (CHD) is the most common birth defect and the leading cause of mortality
in the first year of life with a prevalence of 1% in
live births and 10% in spontaneously aborted fetuses (1). CHD is a disorder mainly characterized by:
a) 90% multifactor disorders, b) 8% chromosomal
and single gene disorders and c) 2% environmental teratogens (2). Among chromosomal disorders
is DiGeorge syndrome (DGS) which is also known
as velocardiofacial syndrome, caused by a submicro-
*Corresponding author: Emina Vukas

Pediatric Clinic, Clinical Centre University of Sarajevo,
Patriotske lige 81, 71000 Sarajevo, Bosnia and Herzegovina
Phone: +387 61 364 347
E-mail: emina.vks@gmail.com
scopic chromosome deletion of band 22q11. It has

malformations attributed to abnormal development
of the pharyngeal arches and pouches. The common
thread among all the organs involved in DiGeorge
anomaly is that their development depends on migration of neural crest cells to the region of pharyngeal pouches.
Clinical features of this syndrome are: congenital
cardiac defects, congenital immunodeficiency secondary to aplasia or hypoplasia of the thymus, and
hypocalcaemia due to small or absent parathyroid
glands, cognitive, behavioral, and psychiatric problems and increased susceptibility to infections (3,4).
One of the most widely cited article estimated that
prevalence rate for DiGeorge syndrome is approximately 1 in 4,000 live births (5,6). In Federation of
Bosnia and Herzegovina in the period between 2008
Submitted Feruary 12, 2014/Accepted March 30, 2014
2014 Senka Mesihovi Dinarevi, Emina Vukas; licensee University of Sarajevo - Faculty of
Health Studies. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
http://www.jhsci.ba
TABLE 1. Findings related to DiGeorge syndrome in the two patients

Features of DGS
Thymic hypoplasia/aplasia
Recurrent infection
Hypoparathyreoidism
Patient 1
broad nose, nostrils in anteversion, wrinkled forehead,
deep-set eyes. Ears were low set, deficient in vertical
diameter and dysplastic
Thymic aplasia
Four episodes of respiratory infection
-
Heart defect
Tetralogia Fallot
Count of Lymphocytes
16.5%
Dysmorphic findings
Patient 2
Thymic aplasia
Interruption of the aortic arch, VSD, DAP,
stenosis of the left pulmonary artery.
16.7%
number of lymphocytes. On X ray of the chest, thymus shadow was not visible. Thymic aplasia was
confirmed during the operation of CHD. Operative
and postoperative course, passed without complications, child was discharged home, recovered, on 10th
day of hospitalization. Three years follow up, child
is in good general condition, body weight 15.2 kg
(p 10), echocardiogram shows unchanged hemodynamic without residual shunt at the ventricular level,
RVOT with PG 28/10 mmHg, without effusion,
thrombus, vegetation, LV EDD (end diastolic diameter), 30 mm, 18 mm ESD (end systolic diameter),
septum and LVPW (left ventricular posterior wall)
5 mm, 40% FS.
2013, there have been two cases of DiGeorge syndrome, prevalence rate is approximately 1:10.000
live births, probably because we do not have the data
from whole country of Bosnia and Herzegovina, as
well as because certain number of this syndrome is
not associated with CHD and remain undiagnosed.
We report two cases of congenital heart disease with
confirmed microdeletion chromosome 22q11.2 by
karyotype and Fluorescence In Situ Hybridization
analysis (FISH).
CASE REPORTS
Case 1.
A ten months old male infant was referred to our

Clinic for operative correction of congenital heart
defect. The child was born as a preterm neonate in
35 week of gestation by vaginal delivery at local hospital, birth weight 2900 grams. He was forth born
child. Other children were normal. There was no
history of antenatal complications. Sucking reflex
was normal. Early psychomotor development was
delayed, he was unable to sit, had recurrent episodes
of respiratory infection, for which he was hospitalized for the treatment. There was no similar problem
in the family in either sides. Physical examination
revealed dysmorphic features, hypotrophic musculature, dyspnea, pallor. His weight was 6.29 kg, p <3,
had broad nose, nostrils in ante version, wrinkled
forehead, deep-set eyes. Ears were low set, deficient
in vertical diameter and dysplastic. Slim and long
fingers on hands and feets. Extensive cardiovascular work up including echocardiography, confirmed
Tetralogy of Fallot. There was no hypocalcemia,
normal count of immunoglobulins and decreased
Case 2.
A seven days old male newborn was admitted in local

hospital in a state of circulatory shock, cardio respiratory decompensated, in severe metabolic acidosis.
After condition has stabilized (intubation, complete
FIGURE 1. Karyotype patient 1

60
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FIGURE 2. FISH for Case 1. Metaphase and interphase cells hybridized with LSI N25 region probe by Visisa, Inc.The absence
of one red signal indicates that the region 22q11.2 microdeletion is present.
On the control examination, nine months after operative procedure, body weight 9.9 kg, vital parameters in the reference values for age. An ultrasound
of the heart hemodynamic unchanged, Ao ascedens
flow in the systole normal. Ao descedens at systole
PG 30 mmHg, LK EDD 23 mm, ESD 13 mm, S
and LVPW 6 mm, FS 40%.
Since the both patients had findings suggestive on
DiGeorge syndrome, karyotype and FISH analysis
was done. Deletion of 22q11.2 region on the q arm
of chromosome 22 has been determined by the LSI
N25 Spectrum Orange/LSI ARSA SpectrumGreen
FISH probe and 90% cells had deletion of 22q11.
Deletion 22q11.2 region, is a microdeletion that
cannot be determined by karyotype analysis (Figure
1.), only by the FISH analysis (Figure 2).
mechanical ventilation, inotropic support), echocardiogram was realized, which confirmed complex
congenital heart anomaly, prostaglandins were administered and the newborn was transferred to the
Pediatric Clinic, Department of Neonatal Intensive
Care Unit for additional diagnostic and surgical
treatment. The child was born as a term neonate by
vaginal delivery at local hospital, birth weight 3130
grams. He was second born child. There was no history of antenatal complications. Sucking reflex was
normal. His weight was 2.89 kg, p<3. Extensive
cardiovascular work up including echocardiography and CT angiography confirmed interruption
of the aortic arch, ventricular septal defect, patent
ductus arteriosus, pulmonary artery dilatation and
stenosis of the left pulmonary artery. There was no
hypocalcaemia. During the operation of CHD thymic aplasia was confirmed. Surgical correction was
uneventful, except the appearance of nodal tachycardia which was successfully treated by medicaments. Postoperatively patient was hemodynamic
stabile, eighth postoperative day control ultrasound
of the heart showed good postoperative result, except pericardial effusion (3 mm), the treatment included non-steroidal anti rheumatic drugs on which
we received a favorable response. Discharged from
the hospital fourteenth postoperative day, recovered.
DISCUSSION
Approximately 7580% of patients with DGS have

congenital heart disease with conotruncal and ventricular septal defects. Various cardial malformations
are seen, particularly affecting the outflow tract (6).
In a series of 545 patients with 22q11 deletions (7),
only 20% had no cardiac defects (ie, based on clinical examination and echocardiography findings).
The most common cardiac anomalies included Te61
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CONCLUSION
tralogy of Fallot (17%), ventricular septal defect

and interrupted aortic arch (14% each), pulmonary
atresia/ventricular septal defect (10%), and truncus
arteriosus (9%), other anomalies included pulmonic
stenosis, atrial septal defect, atrioventricular septal
defect, and transposition of the great arteries. Thymic hypoplasia/aplasia, palate defects and thyroid
and parathyroid abnormalities were described as
well. A congenital heart defect is the main cause of
morbidity and mortality, but the underlying molecular pathobiology is not well understood. Mortality
rate was 8% in a study with 558 patients (7). Most
deaths in this study occurred within 6 months after
birth. Infections due to severe immune deficiency
are the second most common cause of mortality.
According to the current literature, in patients with
Tetralogy of Fallot with/without pulmonary atresia and truncus arteriosus, in spite of the complex
cardiac anatomy, the presence of 22q11.2 deletion
syndrome does not worsen the surgical prognosis.
On the contrary in children with pulmonary atresia with ventricular septal defect and in those with
interrupted aortic arch the association with 22q11.2
deletion syndrome is probably a risk factor for the
operative treatment (8).
In both our patients we had good postoperative outcome, although it was a complex CHD (Tetralogia
Fallot and Interruption aortic arch).
The complex cardiovascular anatomy in association
with depressed immunological status, pulmonary
vascular reactivity, neonatal hypocalcemia, bronchomalacia and broncospasm, laryngeal web, and
tendency to airway bleeding must be considered at
the time of diagnosis and surgical correction. Specific diagnostic, surgical, and perioperative protocols
should be applied in order to provide appropriate
treatment and reduce surgical mortality and morbidity (8).
As for its diagnosis, high-resolution karyotyping has
limitations and is able to identify less than 15% of
affected patients. Fluorescence in situ hybridization
(FISH), which can detect over 90% of the cases, is
considered the gold standard (9). The wide availability of commercial FISH probes has enhanced
the clinicians ability to diagnose and treat the affected children rapidly (10). Identification of these
patients is essential for their adequate management
and genetic counseling.
Our report suggests that the criteria in searching

for microdeletion 22q11.2 should be expanded
and applied in patients with conotruncal and nonconotruncal congenital heart defects and at least one
typical feature of this syndrome (facial dysmorphy,
thymus hypoplasia/aplasia, cleft palate or hypocalcaemia). The phenotype can be extremely variable,
frequently leading to clinical confusion, diagnostic
delay, excess morbidity, early mortality. A multidisciplinary approach is fundamental to ensure that
the patient will be able to attain his or her maximal
potential.
COMPETING INTERESTS
The authors declare that they have no competing

interests.
ACKNOWLEDGMENTS
We would like to thank Department of Pathology

and Cytology,Clinical Center University of Sarajevo
for their technical support.
REFERENCES
1. Kaneko Y, Chevtchik O,Mohl W, et al. Recent advances in genetics of cardiac diseases. Heart View 1999;1(5):152-62.
2. Larson RS, Butler MG: Use of fluorescence in situ hybridization (FISH) in
the diagnosis of DiGeorge sequence and related disease diagnosis. Mol
Pathol. 1995;4(4):274-8.
3. Butts SC. The facial phenotype of the velo-cardio-facial syndrome. Int J
Pediatr Otorhinolaryngol. 2009;73:343350.
4. Cuneo BF. 22q11.2 deletion syndrome: DiGeorge, velocardiofacial, and
conotruncal anomaly face syndromes. Curr Opin Pediatr. 2001;13:465
472.
5. Wilson DI, Burn J, Scambler P, Goodship J. DiGeorge syndrome: part of
CATCH 22. J Med Genet. Oct 1993;30(10):852-6.
6. Oskarsdottir S, Vujic M, Fasth A (2004) Incidence and prevalence of the
22q11 deletion syndrome: a population-based study in Western Sweden.
Arch Dis Child 89: 148151.
7. Ryan AK, Goodship JA, Wilson DI, et al. Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study. J Med Genet. Oct 1997;34(10):798-804.
8. Carotti A, Digilio MC, Piacentini G, Saffirio C, Di Donato RM, Marino B. Cardiac defects and results of cardiac surgery in 22q11.2 deletion syndrome.
DevDisabil Res Rev. 2008;14(1):35-42.
9. Rosa RFM, Zen PRG, Roman T, Graziadio C, Paskulin GA. Sndrome de
deleo 22q11.2: compreendendo o CATCH22. Rev Paul Pediatr. 2009;
27:211-20.
10. Yakut T, Kilic SS, Cil E, Yapici E, Egeli U. FISH investigation of 22q11.2
deletion in patients with immunodeficiency and/or cardiac abnormalities.
Pediatr Surg Int. 2006;22:380383.
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Dragan Stevanovi et al. Journal of Health Sciences 2014;4(1):63-67

CASE REPORT
Open Access
Antiphospholipid syndrome associated with noninfective mitral valve endocarditis: a case report
Dragan Stevanovi*, Denis Maki, Elvira Dambasovi, Amir ehaji, Faruk ustovi,
Nijaz Tucakovi
Department of Internal Medicine, General Hospital Prim.dr Abdulah Naka,Sarajevo, Bosnia and Herzegovina
ABSTRACT
We present a rare case of antiphospholipid syndrome associated with non-infectious thrombotic endocarditis of the mitral valve. The patient was admitted to hospital for examination because of skin lesions
manifested through a discoid skin rash. During the hospitalization antiphospholipid syndrome was diagnosed along with ultrasound verification of vegetations on the mitral valve, including both leaflets, with
moderate to severe mitral regurgitation. Adequate and opportunely introduced therapy led to regression
of all symptoms, including endocarditis of the mitral valve on checkup ultrasound verifications, with a
prevention of arterial and/or venous thrombosis in patient's future.
Keywords: antiphospholipid syndrome, antiphospholipid antibodies, endocarditis.
INTRODUCTION
Antiphospholipid syndrome is a rare autoimmune

disease characterized by antiphospholipid antibodies, clinically manifested in the form of recurrent
arterial and/or venous thrombosis along with recurrent miscarriages. There are 20 or more different
antiphospholipid antibodies which act against negatively charged proteins which bind to phospholipids. The antibodies with most clinical importance
are lupus anticoagulant AB (LA), anticardiolipin
antibodies and anti--2 glycoprotein I antibodies
(a2gp-I). Antiphospholipid antibodies are easily
*Corresponding author: Dragan Stevanovi

Department of Internal Medicine, General Hospital Prim.
dr Abdulah Naka, Kranjevieva 12, 71000 Sarajevo,
Bosnia and Herzegovina; Phone 387 33 285 100
E-mail: dragan.stevanovic@obs.ba
detected by ELISA tests. Even though they represent a heterogeneous group of antibodies, the main
interest remains on those which are closely related
to the clinical manifestations of the disease. Their
most dominant activity is against serum phospholipid binding proteins, initially named co-factors, in
comparison to reaction against phospholipids only.
The most frequent of these proteins is 2 glycoprotein which is bound to negatively charged phospholipids throughout a series of reactions. Physiological role of the 2 glycoprotein is unknown, but it
is assumed that it is a natural anticoagulant in-vivo,
especially because of its ability to bind to the negatively charged phospholipids, inhibiting the activation of the intrinsic pathway of coagulation. It is
considered that 2 glycoprotein is the main target of
the autoimmune antiphospholipid antibodies, but
there are also other phospholipid binding proteins
which play the similar role in the human body, and
2014 Dragan Stevanovi et al.; licensee University of Sarajevo - Faculty of Health Studies.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
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FIGURE 1. Transesophageal echocardiogram on mitral valve during first hospitalization
valve leaflets with verrucae on the ventricular side

of mitral valve along with moderate to severe mitral
regurgitation. Presence of mitral valve endocarditis with mitral regurgitation grade II/III found on
transthoracic echocardiogram (TTE) was confirmed
on transesophageal ultrasound (Figure 1).
Laboratory tests revealed highly positive anti-cardiolipin IgG titer higher than 100, anti-cardiolipin
IgM 23.47, anti 2-glycoprotein IgG also with high
values over 100, anti 2-glycoprotein IgM 3.71. The
tests were repeated after one month: anticardiolipin
IgG higher than 100, anticardiolipin IgM 23.45,
anti 2-glycoprotein IgG 59.42, anti 2-gylcoprotein IgM 4.03. IgG slightly elevated with a value
of 19.10g/l , other immunoglobulins in reference
ranges, C3 slightly decreased 0.889 , C4 in referent
ranges of 0.127 g/l., Anti-streptolysin O (ASTO)
was mildly increased 313 IU, increased erythrocyte sedimentation rate of 34 mm/hour.
Oral anticoagulants were introduced in therapy
with a goal INR values between 2-3, methylprednisolone 1mg/kg and cytostatic therapy. Cyclophosphamide bolus was followed by a pulse therapy with
cyclophosphamide premedication and postmedication, once a month for a period of 6 months, continued by a 3 month scheme, two doses, with gradual
reduction of the dose of methylprednisolone at a
maintenance dose of 8mg per day. Antihypertensive
therapy was continued with carvedilol and enalapril.
On follow up echocardiography the significant regression of verrucae formations were seen on both
they include prothrombin, protein C, protein S and

annexin V (1). Generally the diagnosis of the antiphospholipid syndrome is set when there is at least
one clinical sign (vascular thrombosis and/or spontaneous miscarriage) along with increasing values
of at least one type of antiphospholipid antibodies
such as cardiolipin antibodies (aCL) or lupus anticoagulants antibodies (LA) (2). Patients diagnosed
with antiphospholipid syndrome frequently have
cardiovascular complications. It is most commonly
present as coronary heart disease and non-infectious
endocarditis of the mitral valve, followed by aortic
and tricuspid valve. Described complications occur
in two thirds of all cases (3). We present here a rare
case of antiphospholipid syndrome associated with
non-infectious thrombotic endocarditis of the mitral valve.
CASE REPORT
A 33 year old female patient was admitted to the

hospital because of erythematous skin changes
on her face, behind the right ear, on the left wrist
and on the skin of both knees. The patient complained about fatigue and shortness of breath. She
had a gall bladder resection surgery seven years ago
and she has been diagnosed with an arterial hypertension grade I for 13 years. She is on medication
therapy with carvedilol and enalapril. Two years ago
the patient was diagnosed with cutaneous form of
Discoid lupus erythematosus. Echocardiography on
first admission revealed a thickening of both mitral
64
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FIGURE 2. TTE findings on mitral valve at follow up examination after one year
sions consist of non-infectious warts or thickening

of the leaflets. These two forms can be combined
during the same pathological process and both of
them can be associated with different degrees of
valvular dysfunction. The predominant functional
abnormality is valvular insufficiency while stenosis
is very rare. Mitral valve is the most affected one,
followed by the aortic valve. Valvular dysfunction
usually does not cause clinically manifested valvular disease. The presence of antiphospholipid antibodies increases the future risk for cerebrovascular
complications such as stroke caused by an embolus
from an affected valve. In the treatment of valvular
lesions the most important thing is to establish an
adequate anticoagulation using oral anticoagulant
therapy along with corticosteroids and immunosuppressive agents (4). Antiphospholipid syndrome
may be the cause of recurrent myocardial infarction.
A case report from Snipelisky et al. shows a patient
with antiphospholipid syndrome who had three
myocardial infarctions over a period of two weeks,
who was treated with primary percutaneous coronary intervention with stent implantation in the
first myocardial infarction (5). The most frequent
nonvalvular cardiac manifestation in patients with
antiphospholipid syndrome is pulmonary hypertension (6). According to a follow-up echocardiography
study by Kampolis et al. in patients with antiphospholipid syndrome and systemic lupus erythematosus, disease duration is an independent factor for the
progression of valvular disease, and anticoagulants
could not stop the advancement of the valvular dis-
mitral valve leaflets, with a reduction of valvular

dysfunction and regression of skin changes, which
were the primary reason for patient's hospitalization.
Moderate to severe mitral regurgitation improved to
a moderate to mild form (Figure 2).
On all ultrasound findings systolic (Ejection Fraction 60%) and diastolic function of the left ventricle
was preserved. Also, after the immunosuppressive
therapy in control findings a significant improvement was found: anticardiolipin IgG slightly positive 12.9, anticardiolipin IgM negative 5.0, anti- 2glycoprotein IgG negative 7.8, anti- 2-glycoprotein
IgM 5.96 with normal sedimentation rate of 4mm/
hour.
DISCUSSION
There are primary and secondary antiphospholipid

syndrome, later one occurring in autoimmune diseases, primarily in systemic lupus erythematosus.
There are described cases in which antiphospholipid
syndrome occurs within malignant and infectious
diseases or after certain drug use. The most common
clinical manifestations of this syndrome are thrombosis, which can affect any blood vessel and any
organ in the body. Libman-Sacks endocarditis is an
uninfectious valve damage, associated with autoimmune diseases such as antiphospholipid syndrome
and systemic lupus erythematosus. Echocardiography studies suggest that abnormalities of cardiac
valves are observed in one third of patients affected
by primary antiphospholipid syndrome. Valvular le65
ease or ventricular diastolic dysfunction (7). Sometimes acute myocardial infarction is the first manifestation of antiphospholipid syndrome (8). Although
endocarditis within the antiphospholipid syndrome
is usually mild and asymptomatic, complications
can include an superimposed bacterial infection,
thromboembolic complications and severe regurgitation and/or stenosis which requires cardiac surgery
(9). Venous thrombosis, usually on lower extremities
occur in 55% of cases within this syndrome. Arterial
thrombosis involves brain in 50 % of the cases as
a transient ischemic attack or stroke. Vascular occlusion may occur as a result of embolization from
mitral or aortic valve, and have been reported in 4 %
of cases. For the diagnosis of antiphospholipid syndrome according to "Saporo criteria, patients must
have a vascular thrombosis or spontaneous miscarriage and proven presence of antiphospholipid antibodies, or anticardiolipin antibodies or positive
lupus anticoagulant. Antibodies must be recorded
at least twice at an interval of 6 weeks to distinguish
persistent from transient autoimmune antibodies
responses that can be caused by infectious diseases
or use of certain medications. These criteria have
a 71% sensitivity and a 98% specificity, suggesting that the threshold for making the diagnosis is
high and that most of the cases who are subject to
these criteria definitely have antiphospholipid syndrome (1). Echocardiographic valve abnormalities
are present in about two-thirds of patients with antiphospholipid syndrome, although they are rarely
presented as severe and do not have a very important
clinical relevance. One of the most important complications of the disease are certainly recurrent miscarriages , and the risk of pregnancy loss is increased
from the tenth week of gestation. This is in contrast
with the loss of a pregnancy in the general population, which commonly occurs by the ninth week of
pregnancy (1). A small number of patients have a
so-called catastrophic antiphospholipid syndrome
that some authors also refer to as the "thrombotic
storm" (Catastrophic antiphospholipid syndrome CAPS). In these patients blood vessels from at least
three organic systems are affected, and there is a
high mortality rate. The base of antiphospholipid
therapy is full anticoagulation obtained using oral
anticoagulans, with a preset INR. Based therapy of
antiphospholipid syndrome is full anticoagulation
http://www.jhsci.ba
obtained with oral anticoagulants and targeted INR

values in range from 2 to 3, along with the treatment
of the underlying disease with immunomodulators.
The case report of this patient demonstrated a very
effective treatment strategy with oral anticoagulants,
methylprednisolone and cyclophosphamide, which
led to a significant regression of symptoms with almost complete correction of laboratory results and
significant regression of non-infectious thrombotic
endocarditis of the mitral valve with a notable decrease of dysfunction presented through a regression
of mitral regurgitation.
CONCLUSION
We presented a successful diagnostic protocol that

led to recognition and diagnosis of antiphospholipid syndrome, a rare disease with cardiac complications in the form of noninfectious thrombotic endocarditis of the mitral valve, manifested through
a significant dysfunction of the mitral valve in the
form of moderate to severe mitral regurgitation. Adequate diagnosis and appropriate and timely administered therapy resulted in a significant regression of
the mitral valve endocarditis and prevention of the
possible occurrence of venous or arterial thrombosis
in the patient.
CONFLICT OF INTEREST
None to declare.
REFERENCES
1. Hanly J. Antiphospholipid syndrome: an overview. CMAJ. 2003;

168(13):1675-1682.
2. Ardalan M, Vahedi A. Antiphospholipid syndrome: A disease of protean
face. J Nephropathol. 2013;2(1):81-84.
3. Koniari I, Siminelakis S, Bajkoussis N, Papadopoulos G, Goudevenos J,
Apostolakis A. Antiphospholipid syndrome; its implication in cardiovascular
diseases: a review. 2010;5:101
4. Hojnik M, George J, Ziporen L, Shoenfeld Y. Heart valve involvement
(Libman-Sacks endocarditis) in the antiphospholipid syndrome. Circulation.
1996;93(8):1579-87.
5. Snipelisky D, Stancampiano F, Shapiro B. Antiphospholipid syndrome as a
cause for recurrent myocardial infarction. Acute Card Care. 2013;15(3):802.
6. Pardos-Gea J, Avegliano G, Evangelista A, Vilardell M, Ordi-Ros J. Cardiac manifestations other than valvulopathy in antiphospholipid syndrome:
long-time echocardiography follow-up study. Int J Rheum Dis. 2013. Oct 17
[Epub ahead of print]
7. Kampolis C, Tektonidou M, Moyssakis I, Tzelepis GE, Moutsopoulos H,
Vlachoyiannopulos PG. Evolution of cardiac dysfunction in patients with
antiphosphololipid antibodies and /or antiphosphololipid syndrome: A 10
year follow-up study. Semin Arthritis Rheum. 2014;43(4):558-65.
66
http://www.jhsci.ba
8. Goznales-pacheco H, Eid-Lidt G, Pina-Reyna Y, Amezcua-Guerra LM,

Aldana-Sepuveda N, Martinez-Sanchez C. Acute left main coronary artery
thrombosis as the first manifestation of systemic lupus erythematosus and
catastrophic antiphospholipid syndrome. Am J Emerg Med. 2014;32(2).
9. Bouma W, Klinkenberg T, Van der Horsi I, Wijdh-den Hamer I, Erasmus M,

Bijal M et al. Mitral valve surgery for mitral regurgitation cause by LibmanSacks endocarditis: a report of four cases and a systemic review of the
literature. J Cardiothoracsurg 2010;5:13.
67
Yiit Akn et al. Journal of Health Sciences 2014;4(1):68-71
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CASE REPORT
Open Access
Bilateral Epididymal Cyst in 14 year-old Boy:

a case report
Yiit Akn1, Mehmet Sarac2, Isil Basara3, Selcuk Yucel4, Ahmet Kazez2
1
Department of Urology, Harran University School of Medicine, 63100, Sanliurfa, Turkey. 2Department of Pediatric Surgery,
Firat University School of Medicine, 23042, Elazig, Turkey. 3Department of Radiology, Harput State Hospital, 23050, Elazig,
Turkey. 4Department of Urology, Akdeniz University School of Medicine, 07059, Antalya, Turkey.
ABSTRACT
Bilateral epididymal cysts are rare in childhood. Clinically they may present as acute scrotum and should
be differentiated from other pathologies. Herein, we report bilateral epididymal cysts in a 14-year-old boy.
He was admitted to emergency department with symptoms of acute left scrotum. There was no history
of trauma or infection. Blood analyses, including testis tumor markers were unremarkable. Bilateral epididymal cysts were diagnosed on ultrasonography evaluation. Medical treatment did not stop his scrotal
pain. During scrotal exploration, there was no evidence of testicular torsion or any other pathology. An
excision of cyst was performed. Histopathologically, the cyst wall was lined by columnar epithelia. As a
result of these findings, a pathological diagnosis of epididymal cyst was made. The synchronized evaluation of clinical and ultrasonography findings with an appropriate histopathological evaluation can usually
diagnose this rare pathology.
Keywords: Epididymal cyst, microsurgery, pediatrics.
INTRODUCTION
Scrotal masses can be diagnosed by transillumination or a scrotal ultrasonography examination. At

transillumination these masses may show primarily
fluid, such as a tense hydrocele, or solid, such as testicular tumors. If a much firm intratesticular mass
is palpated, evaluation for lymphoma, leukemia, or
metastatic disease should be performed with examination of the lymph nodes. Children with a non-tender testicular mass and signs of precocious puberty
*Corresponding author: Yigit Akn, MD.
Assistant Professor of Urology
Harran University School of Medicine, 63100 Sanliurfa, Turkey
Tel: +90-506-5334999
E-mail: yigitakin@yahoo.com
should be evaluated for a Leyding cell tumor or less

commonly a Sertoli cell tumors (1, 2). Epididymal
cysts may present as extratesticular masses but they
are usually smooth, round, and characteristically located within the epididymis (2). They cause acute
scrotum which is referred as the new onset of pain,
swelling, and/or tenderness of intrascrotal contents.
Herein, we report that a 14-year-old boy with acute
scrotum. Bilateral epididymal cysts were diagnosed
at emergency deparment.
CASE REPORT
A 14 year-old-boy was admitted emergency outpatient clinic with painless left hemi-scrotal enlargement. There was no history of scrotal trauma or in-
Submitted October 21, 2014/Accepted March 30, 2014
2014 Yiit Akn et al.; licensee University of Sarajevo - Faculty of Health Studies. This is an
http://www.jhsci.ba
FIGURE 1. Ultrasonograhic view of epididymal cyst. a. On the left side, left epididymal cyst size 9x7mm, b. On the right side
right epididymal cyst size 4,5x3mm.
FIGURE 2. Operational and pathological view of cysts a. The arrow and clemp shows epididymal cyst. b. Hispatological findings
are, the low columnar epitelium are spreaded by the wall of cyst, arrow. (HEX400).
fection. At physical examination, in left epididymal

zone a nodular mass was palpated without enlargement and tenderness of the testis. Additionally, there
was a round small cyst palpated in the right epididymis. In scrotal Doppler ultrasonography, there was
no findings of left testicular torsion, and epididymal
cyst was found bilaterally (Figure 1A and 1B).
In laboratory tests, the testicular tumor markers including lactate dehydrogenase (LDH), human chorionic gonadotropin (HCG), and alpha-fetoprotein
(AFP), blood parameters, urine analysis and the
other biochemical tests were within reference ranges.
Non-steroid analgesics were prescribed for scrotal pain and patient was discharged. The same day
later the patient was admitted again at emergency
department with the same symptoms. The day after,
surgical operation was carried out through a scrotal
incision to remove the mass. The paratesticular mass
was found to be a simple epididymal cyst that was
excised intact (Figure 2A). Histological examination
showed the cyst wall was lined by columnar epithelium. As a result of these findings, a pathological
diagnosis of epididymal cyst was made (Figure 2B).
After two months patient referred to pediatric sur69
http://www.jhsci.ba
cally and pathologically from the multicystic/solid

epididymal cystadenomas that occur in von HippelLindau disease (4). Additionally, sometimes spontaneous resolution can occur in pediatric population,
and surgical intervention is rarely needed (5). Medical treatment options are the first line of therapy, as
in our case. Still, the conservative treatment may
sometimes be uneffective and surgery has to be performed to relieve an acute scrotum symptoms.
Epididymal cysts are diagnosed on physical examination. They are palpated as extratesticular masses
but also they are characteristically smooth, round,
and located within the epididymis. For differential
diagnosis, laboratory tests and scrotal ultrasonography should be performed. These tests help us to
make the distinction between epididymal cyst and
testicular cancer. If a epididymal cyst is diagnosed,
there should be no high level of LDH, HCG, and
AFP in the laboratory tests. As radiological, in ultrasonography examination, epididymal cysts appear
simple or minimally complex cysts and they can be
diagnosed easily (5).
The treatment options depend on patients findings. Most epididymal cysts involute with time (6,
7). Conservative treatment options are usually used
for palliation (8). Epididymal cysts are treated when
they cause symptoms like acute scrotum, scrotal
and/or inguinal pain (8). Primary excision is performed to remove the cyst or cysts (8). The incision
is performed through a median raphe or a unilateral transverse scrotal incision to deliver the testis.
Great care has to be taken in children and younger
men of reproductive age because the operation to
remove an epididymal cyst may cause scar tissue to
form and block the outflow duct of the testis. Children need to consider this risk. In the surgery of epididymal cyst in children, microsurgical techniques
can be used by using optical magnification glasses
(5). We used 4 optical magnification glasses during
surgery. Epididymal cysts can be drained with a syringe under local anaesthetic but this is not advised
because epididymal cysts can return and there is a
risk of introducing infection each time the cysts are
needled and also this includes infection risk of cyst.
Sclerotheraphy is the other option for treatment but
there is not enough study that has not been reported
efficiency of this treatment on children (9, 10).
FIGURE 3. Postoperative control scrotal ultrasonogram, the

size of epididymal cyst on the right epididymis is 4,5x3mm.
Upper arrow shows vaz deferens, the other shows cyst.
geon for postoperative check-up. Physical examination showed a round small cyst palpated on the right
epididymis while left hemiscrotum was without any
pathological findigs. A scrotal ultrasonography was
performed which revealed a cyst in the right epididymis measuring 4.5x3 mm. (Figure 3).
There was no cyst in kidneys or other organs in abdomen. Again the tumor markers, the other blood
parameters, urine analysis, and biochemical tests
were unremarkable. The patient did not have any
additional comorbid disease or history of exposure
to diethylstilboestrol, cryptorchidism, cystic fibrosis
or von Hippel-Lindau disease. The follow-up period
of the patient is still ongoing.
DISCUSSION
Unilateral epididymal cysts are common and happen

at all ages. They are fluid filled cysts arising from the
outflow duct of the testis (the epididymis). They are
most often felt as a pea-sized swelling at the top part
of the testis but they can become larger. Sometimes
they cause acute scrotum in children (3). There are
few published reports on bilateral epididymal cysts
in childhood, in the literature. Herein, we reported
a case of bilateral epdidymal cysts.
The pathophysiology of epididymal cysts is still unknown but there are some reports that these cysts
were related to an altered hormonal environment
(1). These lesions are different ultrasonographi70
http://www.jhsci.ba
4. Choyke PL, Glenn GM, Wagner JP, Lubensky IA, Thakore K, Zbar B, Linehan WM, Walther MM. Epididymal cystadenomas in von Hippel-Lindau
disease. Urology. 1997;49:926-31
Although diagnosing epididymal cyst may not represent significant problem, it is important to make
the distinction between epididymal cyst and testicular cancer in childhood. Conservative treatment
options are the first choice of treatment, but when
surgery is needed microsurgical techniques are safe
and effective for epididmal surgery in children.
5. Kauffman EC, Kim HH, Tanrikut C, Goldstein M. Microsurgical spermatocelectomy: technique and outcomes of a novel surgical approach. J Urol.
2011;185:238-42.
6. Homayoon K, Suhre CD, Steinhardt GF. Epididymal cysts in children: natural history. J Urol. 2004;171:1274-6.
7. Chillon Sempere FS, Dominquez Hinarejos C, Serrano Durba A, Estornell
Moragues F, Martinez-Verduch M, Garcia Ibarra F. Epididymal cysts in
childhood. Arch Esp Urol. 2005; 58: 325-8.
REFERENCES
8. Erikci V, Hosgor M, Aksoy N, Okur O, Yildiz M, Dursun A, Demircan Y, Ornek Y, Genisol I. Management of epididymal cyst in childhood. J Pediatr
Surg. 2013;48:2153-6.
1. Agarwal PK, Palmer JS. Testicular and paratesticular neoplasms in prepubertal males. J Urol 2006;176:875-81.
9. Jahnson S, Sandblom D, Holmng S.A randomized trial comparing 2

doses of polidocanol sclerotherapy for hydrocele or spermatocele. J Urol.
2011;186:1319-23.
2. Wein, Kavoussi,Novick, Partin, Peters: Campbells Urology, 10th ed. Philadelphia, PA,Saunders, 2011, p 3060-1.
3. Rioja J, Snchez-Margallo FM, Usn J, Rioja LA. Adult hydrocele and spermatocele. BJU Int. 2011;107:1852-64.
10. Niedzielski J, Miodek M, Krakos. Epididymal cysts in childhood-conservative or surgical approach? Pol Przegl Chir. 2012; 84: 406-10.
71
Journal of Health Sciences 2014;4(1)
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Book Chapter: Blaxter PS, Farnsworth TP. Social health and class
inequalities. In: Carter C, Peel SA, editors. Equalities and inequalities in health. 2nd ed. London: Academic Press; 1976th p. 165-78.
Internet source: HeartCentreOnline. Boca Raton, FL: HeartCentreOnline, Inc.., C2000-2004 [cited 2004 Oct 15]. Available from:
http://www.heartcenteronline.com/
Personal communications and unpublished works should not appear in the references and should be put in parentheses in the text.
Unpublished paper, accepted for publication, may be cited as a reference with the words "in press", next to the name of the journal. All
the references must be verified by the author.
Abbreviations and symbols

Abbreviations should be defined at their first appearance in the
text. Those not internationally recognized should be avoided. Use
of standard abbreviations is recommended. It is necessary to avoid
abbreviations in the title of manuscript and abstract.
Keywords
After the abstract, 3-10 key words or short phrases should be written, that will assist in indexing the article. Whenever possible, use
terms from Medical Subject Headings list of the National Medical
Library (MeSH, NLM). For more information:
(http://www.nlm.nih.gov/mesh/meshhome.html).
Text
The text of the work must be formatted in standard scientific format.
More information can be obtained by downloading templates from the
website of the journal: http://jhsci.ba/information-for-authors.html
Review articles may have a different structure.
The introduction is a concise part of manuscript. It must contain a
description of the problem that this paper deals with, by showing
the problem from the broader context and current situation, moving to specific problem which this paper tries to resolve. At the end
of the introduction it is necessary to clearly point out the purpose,
goals and/or hypothesis this study.
Methods. This section should be brief. The templates that JHSci has
provided on the website have more information about the content
of this chapter.
Results. Give priority to a graphical representation of the results of
studies, whenever applicable. Use subheadings in order to achieve
greater clarity of work. More information can be found in the templates.
Discussion. This section should give meaning to the results obtained, indicate the new discoveries which have been identified,
indicate the results of other studies that have dealt with a similar
problem. Compare your results with other studies and highlight the
differences and novelties in own results. In this chapter the results
should be comprehensively interpreted, analyzed and new knowledge synthesized from the analysis.
Conclusion. Should be brief and contain the most important facts
which have been identified in the paper. Conclusions must arise
from the results obtained during the investigation, and should include the possible application of these results. Both affirmative and
negating conclusions should be stated.
Tables
Tables have to be placed after the references. Each table must be on
a separate page. Tables should NOT be formatted other than simple
borders and no colors.
Table number and title is written above the table. Table gets number
in the order of appearance in the text, with a clear and sufficiently
informative title, i.e. "Table 3. Text table name.... A reference to the
table in text is written in parentheses, i.e. (Table 3). All the abbreviations in the table must be explained in full below the table. It is desirable to give explanations and comments below the table, which
are essential for the presented results to be understood. Display the
statistical measures of variations such as standard deviation and
standard error of the mean, when applicable.
Figures
Figures have to be placed behind the references and tables (if any).
Each figure must be on a separate page. Figures get the titles by the
order of appearance in the text. The title and number are written
below the figure, for example, "Figure 3. Title text When referring to a figure in the manuscript text, number of the figure has to
be written in parentheses, eg (Figure 3). It is essential that the figure
has a clear and informative title and text below the title which explains the presented results with sufficient details. Figure resolution
must be at least 250-300 dpi, JPG or TIFF.
Acknowledgments
In this section you can specify: (a) contributions and authors who
do not meet enough criteria to be authors, such as support from
colleagues or heads of institutions, (b) thanks for technical assistance, (c) thanks for material or financial assistance, stating the
character of that assistance.
Units of Measure
Measures of length, weight and volume should be written in metric units (meter, kilogram, liter). Hematological and biochemical
parameters should be expressed in metric units according to the
International System of Units (SI).
Statement on Conflict of Interest

Authors must identify all sources of funding of their studies and any
financial aid (including obtaining a salary, pay, etc.) by the institu-
73
Journal of Health Sciences 2014;4(1)
http://www.jhsci.ba
UPUTSTVO AUTORIMA
Upute i smjernice autorima za pripremu i predaju rukopisa u Journal of Health Sciences
Ciljevi i okvir asopisa
ako autori ele predstaviti rukopis, pismo ili dijelove koji ne mogu
biti poslani elektronski, ili je to zatraeno od urednitva. Za autore
koji nemaju mogunost elktronskog slanja rada, potrebno je poslati
potom jedan primjerak rada, zajedno s elektronskom verzijom na
CD-u ili DVD-u na sljedeu adresu: za Journal of Health Sciences,
Fakultet zdravstvenih studija Univerziteta u Sarajevu, 71000 Sarajevo, Bolnika 25, Bosna i Hercegovina.
The Journal of Health Sciences (JHSci) je internacionalni asopis

na engleskom jeziku, koji objavljuje orginalne radove iz oblasti fizikalne terapije, medicinsko-laboratorijske dijagnostike, radioloke
tehnike, sanitarnog inenjerstva, zdravlja i ekologije, zdravstvene
njege i terapije, te drugih srodnih oblasti.
Pravila redakcije
Vrste znanstvenih radova koje se mogu poslati za objavljivanje

u JHS
Autorstvo
Svi autori morati potpisati formular za podnoenje rada (Manuscript Submission form). Potrebno je da svi autori potpisom potvrde
da: su zadovoljili kriterije za autorstvo u radu, utvreno od strane
International Committee of Medical Journal Editors; vjeruju da
rukopis predstavlja poteni rad i da su u mogunosti potvrditi valjanost navedenih rezultata. Autori su odgovorni za sve navode i
stavove u njihovim radovima. Vie informacija se moe dobiti na
(http://bmj.com/cgi/collection/authorship).
Orginalni radovi: orginalne laboratorijske eksperimentalne i klinike studije ne bi trebao prelaziti 4500 ukljuujui tabele i reference.
Prikaz sluajeva: prezentacije klinikih sluajeva koji mogu sugerisati kreiranje nove radne hipoteze, uz prikaz odgovarajue literature. Tekst ne bi trebao prelaziti 2400 rijei.
Pregledni lanci: lanci afirmiranih znanstvenika, pozvanih da ih
napiu za asopis. Redakcija e, takoer, razmatrati i samostalne
aplikacije.
Uvodnici: lanci ili kratki uvodniki komentari koji predstavljaju
miljenja prepoznatih lidera u medicinskim istraivanjima.
Plagijarizam ili dupliciranje objavljenog rada

Od autora se zahtjeva da svojim potpisom potvrde da u momentu
podnoenja rad nije objavljen u sadanjem obliku ili bitno slinom
obliku (u tampanom ili elektronskom obliku, ukljuujui i na web
stranici), da nije prihvaen za objavljivanje u drugom asopisu ili
razmatran za objavljivanje u drugom asopisu. Meunarodni odbor urednika medicinskih asopisa dao je detaljno objanjenje ta
jeste, a ta nije duplikat (www.icmje.org). Vie informacija moe se
nai i na stranici www.jhsci.ba.
Podnoenje rada za objavljivanje

Rad koji se alje u JHSci mora biti u skladu sa propozicijama o sadraju, izgledu i kvalitetu, koje je urnal propisao u ovim instrukcijama za autore i na web stranici urnala, www.jhsci.ba. Propozicije
o sadraju, izgledu i kvalitetu naunog rada u skladu su sa meunarodnim propozicijama i preporukama datim od strane International Committee of Medical Journal Editors. Uniform Requirements
for Manuscripts Submitted to Biomedical Journals New Engl J
Med 1997, 336:309315 (www.icmje.org), te preporuka meunarodnih radnih grupa za standardizaciju izgleda i kvaliteta naunih
radova: STROBE (www.strobe-statement.org) , CONSORT (www.
consort-statement.org), STARD (www.stard-statement.org) i drugih.
Formular saglasnosti bolesnika

Zatita prava pacijenta na privatnost je od iznimnog znaaja. Autori trebaju, ako redakcija zahtjeva, poslati kopije formulara Suglasnosti bolesnika iz kojih se jasno vidi da bolesnici ili drugi subjekti
eksperimenata daju doputenje za objavljivanje fotografija i drugih
materijala koji bi ih identificirali. Ako autori nemaju potrebnu saglasnost za istraivanje, moraju je dobiti ili iskljuiti podatke koji
identificiraju subjekte, a za koje nisu dobili saglasnost.
Predloci
JHSci je pripremio predloke (engl. template) za izgled i sadraj
naunog rada. Predloci sadre sve neophodne podnaslove i obogaeni su uputama o sadraju svakog poglavlja naunog rada, te e
autorima znatno olakati proces pisanja rada. JHSci preporuuje
koritenje predloaka za pisanje naunih radova koji se nalaze na
web stranici urnala www.jhsci.ba u dijelu Information for authors.
Odobrenje Etikog komiteta

Autori moraju u formularu za podnoenje rada i u dijelu rada
Metode jasno navesti da su studije koje su proveli na humanim
subjektima, odnosno pacijentima, odobrene od strane odgovoarajueg etikog komiteta. Vie informacija moete nai u najnovijoj verziji Helsinke deklaracije (http://www.wma.net/e/policy/
b3.htm). Isto tako, autori moraju potvrditi da su eksperimenti koji
ukljuuju ivotinje provedeni u skladu sa etikim standardima.
Pismo za podnoenje rada

Svi autori rada moraju potpisati formular za podnoenje rada. On
sadri odobrenje za publiciranje poslanog rada, izjavu o sukobu
interesa, izjavu potivanju etikih principa u istraivanju i izjavu o
prijenosu autorskih prava na JHSci. Ovaj formular se mora preuzeti
sa web stranice www.jhsci.ba u dijelu Information for authors, te
odtampati, popuniti i skenirati. Ukoliko se skeniranjem dobiju dva
ili tri fajla, moraju se pretvoriti u jedan ZIP fajl.
Izjava o sukobu interesa

Od autora se zahtjeva da navedu sve izvore finansijske pomoi koje
su dobili za istraivanje (grantovi za projekte, ili drugi izvori finansiranja). Ako ste sigurni da nema sukoba interesa, onda to i navedite kratko. Za vie informacija pogledajte uvodnik u British Medical
Journal, 'Beyond conflict of interest' (http://bmj.com/cgi/content/
short/317/7154/291).
Slanje rada
Vri se iskljuivo preko web stranice www.jhsci.ba preko predvienog web formulara. Web formular sadri etiri stranice na kojima
se nalazi: 1. popis stavki koje treba ostvariti prije podnoenja rada;
2. informacije o autoru za korespondenciju; 3. informacije o naunom radu; 4. dio za slanje fajlova. U web formularu autori su
duni ispravno popuniti informacije, unijeti ispravnu e-mail adresu za korespondenciju, te poslati 2 fajla: 1. Pismo za podnoenje
rada; 2. Nauni rad. NIJE POTREBNO slati tampanu verziju, osim
Izdavaka prava
U okviru Pisma za podnoenje rada od autora se zahtjeva da prenesu izdavaka prava na Fakultet zdravstvenih studija. Prijenos izdavakih prava postaje punovaan kada i ako rad bude prihvaen
za publiciranje. ira javnost ima prava reproducirati sadraj ili listu
lanaka, ukljuujui abstrakte, za internu upotrebu u svojim institucijama. Saglasnost izdavaa je potrebna za prodaju ili distribuciju
van institucije i za druge aktivnosti koje proizilaze iz distribucije,
ukljuujui kompilacije ili prijevode. Ukoliko se zatieni materijali
74
Upute i smjernice autorima za pripremu i predaju rukopisa u Journal of Health Sciences
koriste, autori moraju dobiti pismenu dozvolu izdavaa i navesti

izvor, odnosno referencu u lanku.
materijala u radu, ili koji bi mogli uticati na nepristranost studije. Ako ste sigurni da ne postoji sukob interesa, navedite to u radu.
Jo informacija se moe nai ovdje: (http://bmj.com/cgi/content/
short/317/7154/291).
Formatiranje (izgled) rada

Predloci (engl. template) za pisanje radova
JHSci je na svojoj web stranici www.jhsci.ba dao predloke (engl.
Template) prema kojima treba formatirati radove. Predloci, takoer, sadre i upute preuzete od strane radnih grupa za standardiziranje formata u pisanju naunih radova i objektivno i potpuno
prikazivanje rezultata studija. Vie informacija o strukturi naunih
radova moe se nai na web stranici www.jhsci.ba i na web stranicama radnih grupa: www.consort-statement.org, www.strobe-statement.org, www.stard-statement.org, i drugih. Predloci se mogu
preuzeti na sljedeem linku: http://jhsci.ba/information-for-authors.html
Reference
Reference se trebaju numerisati prema redoslijedu pojavljivanja u
radu. U tekstu, reference je potrebno navesti u zagradama, npr. (12).
Kada rad koji citirate ima do 6 autora, navesti sve autore. Ukoliko
je 7 ili vie autora, navesti samo provih 6 i dodati et al. Reference
moraju ukljuivati puni naziv i izvor informacija (Vancouver style).
Imena urnala trebaju biti skraena kao na PubMedu. http://www.
ncbi.nlm.nih.gov/journals
Primjeri referenci:
Standardni rad: Meneton P, Jeunemaitre X, de Wardener HE,
MacGregor GA. Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev.
2005;85(2):679-715
Vie od 6 autora: Hallal AH, Amortegui JD, Jeroukhimov IM, Casillas J, Schulman CI, Manning RJ, et al. Magnetic resonance cholangiopancreatography accurately detects common bile duct stones in
resolving gallstone pancreatitis. J Am Coll Surg. 2005;200(6):86975.
Knjige: Jenkins PF. Making sense of the chest x-ray: a hands-on
guide. New York: Oxford University Press; 2005. 194 p.
Poglavlje u knjizi: Blaxter PS, Farnsworth TP. Social health and
class inequalities. In: Carter C, Peel JR, editors. Equalities and
inequalities in health. 2nd ed. London: Academic Press; 1976. p.
165-78.
Internet lokacija: HeartCentreOnline. Boca Raton, FL: HeartCentreOnline, Inc.; c2000-2004 [cited 2004 Oct 15]. Available from:
http://www.heartcenteronline.com/
Osobne komunikacije i nepublicirani radovi ne bi se trebali nai u
referencama ve biti navedeni u zagradama u tekstu. Neobjavljeni
radovi, prihvaeni za publiciranje mogu se navesti kao referenca sa
rijeima U tampi (engl. In press), pored imena urnala. Reference moraju biti provjerene od strane autora.
Skraenice i simboli
Skraenice se moraju definisati prilikom njihovog prvog pojavljivanja u tesktu. One koje nisu internacionalno i generalno prihvaene
trebaju se izbjegavati. Koristiti standardne skraenice. Potrebno je
izbjegavati skraenice u naslovu rada i u saetku.
Kljune rijei
Nakon abstrakta treba staviti 3-10 kljunih rijei ili kratkih fraza
koje e pomoi u indeksiranju rada. Uvijek kada je to mogue, treba koristiti termine iz Medical Subject Headings liste Nacionalne
Medicinske Bibiloteke (MeSH, NLM). Vie informacija na:
(http://www.nlm.nih.gov/mesh/meshhome.html).
Tekst rada
Tekst rada mora biti standardnog naunog formata. Vie informacija dobiete preuzimanjem predloaka sa web stranice urnala:
http://jhsci.ba/information-for-authors.html
Pregledni lanci mogu imati drugaiju strukturu.
Uvod je koncizan dio rada. U njemu se predstavlja problem kojim
se rad bavi i to kreui od ireg konteksta problema i trenutnog
stanja i dosadanjih dostignua u vezi konkrtnog problema, prema
specifinom problemu koji e obraditi ova studija. Na kraju uvoda
je potrebno jasno istaknuti svrhu, ciljeve i/ili hipoteze ove studije.
Metode. Ovaj dio ne treba biti kratak. U predlocima koje je JHSci dao na web stranici nalazi se vie informacija o sadraju ovog
poglavlja.
Rezultati. Dati prednost grafikom prikazu rezultata studije u odnosu na tabelarni, kada je god to primjenjivo. Koristiti podnaslove
radi postizanja vee jasnoe radova. Vie informacija nai u predlocima.
Diskusija. U ovoj sekciji treba dati smisao dobivenim rezultatima,
ukazati na nova otkria do kojih se dolo, ukazati na rezultate drugih studija koje su se bavile slinim problemom. Uporediti svoje
rezultate sa drugim studijama i naglasiti razlike i novine u svojim
rezultatima. U ovom poglavlju treba interpretirati, sveobuhvatno
sagledati dobijene rezultate, te sintetizirati novo znanje iz analize.
Zakljuak. Treba da bude kratak i da sadri najbitnije injenice do
kojih se dolo u radu. Navodi se zakljuak, odnosno zakljuci koji
proizilaze iz rezultata dobivenih tokom istraivanja; treba navesti
eventualnu primjenu navedenih ispitivanja. Treba navesti i afirmativne i negirajue zakljuke.
Tabele
Tabele se moraju staviti iza referenci. Svaka tabela mora biti na posebnoj stranici. Tabele NE TREBA grafiki ureivati.
Broj tabele i njen naziv pie se IZNAD tabele. Tabela dobija broj
prema redoslijedu pojavljivanja u tekstu, a naziv treba biti jasan i
dovoljno opisan da je jasno ta tabela prikazuje. npr Table 3. Tekst
naziva tabele..... U radu prilikom pozivanja na tabelu treba napisati
broj tabele u zagradi, npr. (Table 3). Za skraenice u tabeli potrebno
je dati puni naziv ispod tabele. Poeljno je ispod tabele dati objanjenja i komentar, koji su neophodni da se rezultati u tabeli mogu
razumjeti. Prikazati statistike mjere varijacije, kao to je standardna devijacija i standardna greka sredine, gdje je primjenjivo.
Slike
Slike staviti iza referenci i tabela (ako postoje). Svaka slika mora biti
na posebnoj stranici. Slika dobija broj prema redoslijedu pojavljivanja u tekstu. Naziv i broj se piu ISPOD slike, npr. Slika 3. Tekst
naziva slike... U radu, prilikom pozivanja na sliku treba napisati
broj slike u zagradi, npr (Slika 3). Neophodno je da slika ima jasan
i indikativan naziv, a u tekstu ipod slike objasniti sliku i rezultat
koji ona prikazuje, sa dovoljno detalja da ona moe biti jasna bez
pretrage teksta koji je objanjava u radu. Slika mora biti kvaliteta
najmanje 250-300 dpi, formata JPG, TIFF ili BMP.
Zahvala
U ovom dijelu se mogu navesti: (a) doprinosi i autori koji ne zadovoljavaju dovoljno kriterija da budu autori, kao npr. podrka kolega
ili efova institucija; (b) zahvala za tehniku pomo; (c) zahvala za
materijalnu ili finansijsku pomo, obrazlaui karakter te pomoi.
Jedinice mjere
Mjere duine, teine i volumena trebaju se pisati u metrikim jedinicama (meter, kilogram, liter). Hematoloki i biohemijski parametri se trebaju izraavati u metrikim jedinicama prema International System of Units (SI).
Izjava o sukobu interesa

Autori moraju navesti sve izvore finasiranja svoje studije i bilo koju
finansijsku potporu (ukljuujui dobijanje plae, honorara, i drugo) od strane institucija iji finansijski interesi mogu zavisiti od
75

JHSCI 2014 v4 I1 April

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JHSCI 2014 v4 I1 April

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UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES

UNIVERZITET U SARAJEVU FAKULTET ZDRAVSTVENIH STUDIJA

Journal of Health Sciences

Dijana Avdi (BiH)

Mirza Dili (BiH)

Ismet Gavrankapetanovi (BiH)

Demal Pecar (BiH)

Sebija Izetbegovi (BiH)

Slobodan Loga (BIH)

Aida Rudi (BiH)

Senka Mesihovi-Dinarevi (BiH)

Ljerka Ostoji (BiH)

Isabelle Rishard (F)

Yigit Akin (TR)

Fatima Jusupovi (BiH)

Renata Dobrila-Dintinjana (CRO)

Kasim Bajrovi (BiH)

Mirsada Huki (BiH)

Antiphospholipid syndrome associated with non-infective

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

Journal of Health Sciences

Lymphangiogenesis in breast carcinoma is present

tion of metastases. Spread to axillary lymph nodes

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

TABLE 1. Clinicopathological characteristics of the studied

lymphatic markers that have allowed observation

Seventy-five cases of invasive carcinoma (IC) of the

Double-labeling immunohistochemical staining. Fourmicron-thick paraffin sections were taken on charged

slides, deparaffinized in xylene, and hydrated with

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

of positive cells versus intensity of staining) and a

The fractions of proliferating lymphatic endothelial

Statistical analysis was performed using SPSS for

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

ing vessels entrapped within the tumor (Figure

Benign breast samples

Lymphatic vessels were identified within invasive

FIGURE 3. LECP show correlation with histological grade of

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

rent study, lymphangiogenesis was assessed by both

FIGURE 4. Axillary lymph node metastasis showed strong

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

viding lymphatic endothelial cells (26%) and some

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

The authors declare no conflict of interest for this

(6) Fox SB, Harris AL Histological quantitation of tumour angiogenesis. Apmis

(26) Dhakal HP, Bassarova A, Naume B, et al. Breast carcinoma vascularity: a

(27) Dadras SS, Paul T, Bertoncini J, et al. Tumor lymphangiogenesis: a novel

Mirsad Dori et al. Journal of Health Sciences 2014;4(1):4-11

tastasis in Primary Breast Cancers. Asian Pacific J Cancer Prev. 2013;14

(29) Kanngurn S, Thongsuksai P, Chewatanakornkul S. Chalkley Microvessel

Lejla Meali Journal of Health Sciences 2014;4(1):12-18

Journal of Health Sciences

Correlation of Body Mass Index and Waist Hip

Menopausal transition is a period in life of a woman,

Lejla Meali Journal of Health Sciences 2014;4(1):12-18

women (1). Hormonal profile is changing during

250 000 patients, shows that the patients with BMI

This prospective, comparative study was conducted

Lejla Meali Journal of Health Sciences 2014;4(1):12-18

TABLE 1. Main features of assessees1

was taken, after which the blood was centrifuged,

years), and the assessees in postmenopause were four